Tendon-inducing compositions

ABSTRACT

Compositions of proteins with tendon/ligament-like tissue inducing activity are disclosed. The compositions are useful in the treatment of tendinitis and tendon or ligament defects and in related tissue repair.

RELATED APPLICATIONS

[0001] The present invention is a continuation-in-part of applicationSer. No. 08/217,780, filed Mar. 25, 1994, 08/164,103, filed on Dec. 7,1993 and 08/333,576, filed on Nov. 2, 1994.

FIELD OF THE INVENTION

[0002] The present invention relates to a novel family of purifiedproteins, and compositions containing such proteins, which compositionsare useful for the induction of tendon/ligament-like tissue formation,wound healing and ligament and other tissue repair. These proteins mayalso be used in compositions for augmenting the activity of bonemorphogenetic proteins.

BACKGROUND OF THE INVENTION

[0003] The search for the molecule or molecules responsible forformation of bone, cartilage, tendon and other tissues present in boneand other tissue extracts has led to the discovery of a novel set ofmolecules called the Bone Morphogenetic Proteins (BMPs). The structuresof several proteins, designated BMP-1 through BMP-11, have previouslybeen elucidated. The unique inductive activities of these proteins,along with their presence in bone, suggests that they are importantregulators of bone repair processes, and may be involved in the normalmaintenance of bone tissue. There is a need to identify additionalproteins which play a role in forming other vital tissues. The presentinvention relates to the identification of a family of proteins, whichhave tendon/ligament-like tissue inducing activity, and which are usefulin compositions for the induction of tendon/ligament-like tissueformation and repair.

SUMMARY OF THE INVENTION

[0004] In one embodiment, the present invention comprises DNA moleculesencoding a tendon/ligament-like inducing protein which the inventorshave named V1-1. This novel protein is now called BMP-12. The presentinvention also includes DNA molecules encoding BMP-12 related proteins.

[0005] BMP-12 related proteins are a subset of the BMP/TGF-β/Vg-1 familyof proteins, including BMP-12 and VL-1, which are defined astendon/ligament-like tissue inducing proteins encoded by DNA sequenceswhich are cloned and identified, e.g., using PCR, using BMP-12 specificprimers, such as primers #6 and #7 described below, with reducedstringency conditions. It is preferred that the DNA sequences encodingBMP-12 related proteins share at least about 80% homology at the aminoacid level from amino acids with amino acids #3 to #103 of SEQ ID NO:1.

[0006] The DNA molecules preferably have a DNA sequence encoding theBMP-12 protein, the sequence of which is provided in SEQ ID NO:1, or aBMP-12 related protein as further described herein. Both the BMP-12protein and BMP-12 related proteins are characterized by the ability toinduce the formation of tendon/ligament-like tissue in the assaydescribed in the examples.

[0007] The DNA molecules of the invention preferably comprise a DNAsequence, as described in SEQUENCE ID NO:1; more preferably nucleotides#496 to #882, #571 to #882 or #577 to #882 of SEQ ID NO:1; or DNAsequences which hybridize to the above under stringent hybridizationconditions and encode a protein which exhibits the ability to formtendon/ligament-like tissue. The DNA molecules of the invention may alsocomprise a DNA sequence as described in SEQ ID NO:25; more preferablynucleotides #604 or #658 to #964 of SEQ ID NO:25.

[0008] The DNA molecules of the invention also include DNA moleculescomprising a DNA sequence encoding a BMP-12 related protein with theamino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:26, as well asnaturally occurring allelic sequences and equivalent degenerative codonsequences of SEQ ID NO:2 or SEQ ID NO:26. Preferably, the DNA sequenceof the present invention encodes amino acids #-25 to #104, #1 to #104 or#3 to #103 of SEQ ID NO:2; or amino acids #1 to #120 or #19 to #120 ofSEQ ID NO:26. The DNA sequence may comprise, in a 5′ to 3′ direction,nucleotides encoding a propeptide, and nucleotides encoding for aminoacids #-25 to #104, #1 to #104 or #3 to #103 of SEQ ID NO:2; or aminoacids #1 to #120 or #19 to #120 of SEQ ID NO:26 The propeptide useful inthe above embodiment is preferably selected from the group consisting ofnative BMP-12 propeptide and a protein propeptide from a differentmember of the TGF-B superfamily or BMP family. The invention furthercomprises DNA sequences which hybridize to the above DNA sequences understringent hybridization conditions and encode a BMP-12 related proteinwhich exhibits the ability to induce formation of tendon/ligament-liketissue.

[0009] In other embodiments, the present invention-comprises host cellsand vectors which comprise a DNA molecule encoding the BMP-12 protein,or a BMP-12 related protein. The host cells and vectors may furthercomprise the coding sequence in operative association with an expressioncontrol sequence therefor.

[0010] In another embodiment, the present invention comprises a methodfor producing a purified BMP-12 related protein, said method comprisingthe steps of culturing a host cell transformed with the above DNAmolecule or vector comprising a nucleotide sequence encoding a BMP-12related protein; and (b) recovering and purifying said BMP-12 relatedprotein from the culture medium. In a preferred embodiment, the methodcomprises (a) culturing a cell transformed with a DNA moleculecomprising the nucleotide sequence from nucleotide #496, #571 or #577 to#879 or #882 as shown in SEQ ID NO:1; or the nucleotide sequence from#604 or #658 to #963 of SEQ ID NO:25; and

[0011] (b) recovering and purifying from said culture medium a proteincomprising the amino acid sequence from amino acid #-25, #1 or #3 toamino acid #103 or #104 as shown in SEQ ID NO:2; or from amino acid #1or #19 to amino acid #120 as shown in SEQ ID NO:26. The presentinvention also includes a purified protein produced by the abovemethods.

[0012] The present invention further comprises purified BMP-12 relatedprotein characterized by the ability to induce the formation oftendon/ligament-like tissue. The BMP-12 related polypeptides preferablycomprise an amino acid sequence as shown in SEQ ID NO:2. The polypeptidemore preferably comprise amino acids #-25, #1 or #3 to #103 or #104 asset forth in SEQ ID NO:2; or amino acids #1 or #19 to #120 as set forthin SEQ ID NO:26. In a preferred embodiment, the purified polypeptide maybe in the form of a dimer comprised of two subunits, each with the aminoacid sequence of SEQ ID NO:2.

[0013] In another embodiment, the present invention comprisescompositions comprising an effective amount of the above-describedBMP-12 related proteins. In the compositions, the protein may be admixedwith a pharmaceutically acceptable vehicle.

[0014] The invention also includes methods for tendon/ligament-liketissue healing and tissue repair, for treating tendinitis, or othertendon or ligament defects, and for inducing tendon/ligament-like tissueformation in a patient in need of same, comprising administering to saidpatient an effective amount of the above composition.

[0015] Other embodiments include chimeric DNA molecules comprising a DNAsequence encoding a propeptide from a member of the TGF-β superfamily ofproteins linked in correct reading frame to a DNA sequence encoding aBMP-12 related polypeptide. One suitable propeptide is the propeptidefrom BMP-2. The invention also includes heterodimeric protein moleculescomprising one monomer having the amino acid sequence shown in SEQ IDNO:2, and one monomer having the amino acid sequence of another proteinof the TGF-β subfamily.

[0016] Finally, the present invention comprises methods for inducingtendon/ligament-like tissue formation in a patient in need of samecomprising administering to said patient an effective amount of acomposition comprising a protein which exhibits the ability to induceformation of tendon/ligament-like tissue, said protein having an aminoacid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:26. Theamino acid sequences are more preferably one of the following: (a) aminoacids #-25, #1 or #3 to #103 or #104 of SEQ ID NO:2; (b) amino acids #1or #19 to #119 or #120 of SEQ ID NO:4; (c) amino acids #1 or #19 to #119or #120 of SEQ ID NO:26; (d) mutants and/or variants of (a), (b) or (c)which exhibit the ability to form tendon and/or ligament. In otherembodiments of the above method, the protein is encoded by a DNAsequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:25, more preferablyone of the following: (a) nucleotides #496, #571 or #577 to #879 or #882of SEQ ID NO:1; (b) nucleotides #845 or #899 to #1201 or #1204 of SEQ IDNO:3; (c) nucleotides #605 or #659 to #961 or #964 of SEQ ID NO:25; and(d) sequences which hybridize to (a) or (b) under stringenthybridization conditions and encode a protein which exhibits the abilityto form tendon/ligament-like tissue.

[0017] Description of the Sequences

[0018] SEQ ID NO:1 is the nucleotide sequence encoding the human BMP-12.

[0019] SEQ ID NO:2 is the amino acid sequence comprising the maturehuman BMP-12 polypeptide.

[0020] SEQ ID NO:3 is the nucleotide sequence encoding the protein MP52.

[0021] SEQ ID NO:4 is the amino acid sequence, comprising the matureMP52 polypeptide.

[0022] SEQ ID NO:5 is the nucleotide sequence of a specificallyamplified portion of the human BMP-12 encoding sequence.

[0023] SEQ ID NO:6 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:5.

[0024] SEQ ID NO:7 is the nucleotide sequence of a specificallyamplified portion of the human VL-1 encoding sequence.

[0025] SEQ ID NO:8 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:7.

[0026] SEQ ID NO:9 is the nucleotide sequence of the plasmid pALV1-781,used for expression of BMP-12 in E. coli.

[0027] SEQ ID NO:10 is the nucleotide sequence of a fragment of themurine clone, mV1.

[0028] SEQ ID NO:11 is the amino acid sequence of a fragment of themurine protein encoded by mV1.

[0029] SEQ ID NO:12 is the nucleotide sequence of a fragment of themurine clone, mV2.

[0030] SEQ ID NO:13 is the amino acid sequence of a fragment of themurine protein encoded by mV2.

[0031] SEQ ID NO:14 is the nucleotide sequence of a fragment of themurine clone, mV9.

[0032] SEQ ID NO:15 is the amino acid sequence of a fragment of themurine protein encoded by mV9.

[0033] SEQ ID NO:16 is the amino acid sequence of a BMP/TGF-β/Vg-1protein consensus sequence The first Xaa represents either Gln or Asn;the second Xaa represents either Val or Ile.

[0034] SEQ ID NO:17 is the nucleotide sequence of oligonucleotide #1.

[0035] SEQ ID NO:18 is the amino acid sequence of a BMP/TGF-β/Vg-1protein consensus sequence. The Xaa represents either Val or Leu.

[0036] SEQ ID NO:19 is the nucleotide sequence of oligonucleotide #2.

[0037] SEQ ID NO:20 is the nucleotide sequence of oligonucleotide #3.

[0038] SEQ ID NO:21 is the nucleotide sequence of oligonucleotide #4.

[0039] SEQ ID NO:22 is the nucleotide sequence of oligonucleotide #5.

[0040] SEQ ID NO:23 is the nucleotide sequence of oligonucleotide #6.

[0041] SEQ ID NO:24 is the nucleotide sequence of oligonucleotide #7.

[0042] SEQ ID NO:25 is the nucleotide sequence of the human VL-1(BMP-13) encoding sequence.

[0043] SEQ ID NO:26 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:25.

[0044] SEQ ID NO:27 is the nucleotide sequence encoding a fusion ofBMP-2 propeptide and the mature coding sequence of BMP-12.

[0045] SEQ ID NO:28 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:27.

[0046] SEQ ID NO:29 is the nucleotide sequence encoding the murine mV1protein. X01 is Val, Ala, Glu or Gly; X02 is Ser, Pro Thr or Ala; X03 isSer or Arg; X04 is Leu, Pro, Gln or Arg; X05 is Cys or Trp; X06 is Val,Ala, Asp or Gly; X07 is Val, Ala, Glu or Gly; X08 is Gln, Lys or Glu.

[0047] SEQ ID NO:30 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:29. X01 through X08 are the same as in SEQ IDNO:29.

[0048] SEQ ID NO:31 is the nucleotide sequence encoding the murine mV2protein. X01 is Pro or Thr; X02 is Val.

[0049] SEQ ID NO:32 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:31. X01 and X02 are the same as in SEQ ID NO:31.

[0050] SEQ ID NO:33 is the nucleotide sequence encoding human BMP-12protein.

[0051] SEQ ID NO:34 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:33.

[0052] SEQ ID NO:35 is the nucleotide sequence of oligonucleotide #8.

BRIEF DESCRIPTION OF THE FIGURES

[0053]FIG. 1 is a comparison of the human BMP-12 and human MP52sequences.

DETAILED DESCRIPTION OF THE INVENTION

[0054] The DNA sequences of the present invention are useful forproducing proteins which induce the formation of tendon/ligament-liketissue, as described further below. The DNA sequences of the presentinvention are further useful for isolating and cloning further DNAsequences encoding BMP-12 related proteins with similar activity. TheseBMP-12 related proteins may be homologues from other species, or may berelated proteins within the same species.

[0055] Still, a further aspect of the invention are DNA sequences codingfor expression of a tendon/ligament-like tissue inducing protein. Suchsequences include the sequence of nucleotides in a 5′ to 3′ directionillustrated in SEQ ID NO:1 or SEQ ID NO:25, DNA sequences which, but forthe degeneracy of the genetic code, are identical to the DNA sequenceSEQ ID NO:1 or 25, and encode the protein of SEQ ID NO:2 or 26. Furtherincluded in the present invention are DNA sequences which hybridizeunder stringent conditions with the DNA sequence of SEQ ID NO:1 or 25and encode a protein having the ability to induce the formation oftendon or ligament. Preferred DNA sequences include those whichhybridize under stringent conditions as described in Maniatis et al,Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory(1982), pages 387 to 389. Finally, allelic or other variations of thesequences of SEQ ID NO:1 or 25, whether such nucleotide changes resultin changes in the peptide sequence or not, but where the peptidesequence still has tendon/ligament-like tissue inducing activity, arealso included in the present invention.

[0056] The human BMP-12 DNA sequence (SEQ ID NO:1) and amino acidsequence (SEQ ID NO:2) are set forth in the Sequence Listings. Anotherprotein that is useful for the compositions and methods of the presentinvention is VL-1. VL-1 is a BMP-12 related protein which was clonedusing sequences from BMP-12. The inventors have now designated VL-1 asBMP-13. A partial DNA sequence of VL-1 (SEQ ID NO:7) and the encodedamino acid sequence (SEQ ID NO:8); as well as a DNA sequence encodingthe mature VL-1 (SEQ ID NO:25) and the encoded amino acid sequence (SEQID NO:26) are set forth in the Sequence Listings. Although furtherdescriptions are made with reference to the BMP-12 sequence of SEQ IDNO:1 and 2, it will be recognized that the invention includes similarmodifications and improvements which may be made to other BMP-12 relatedsequences, such as the VL-1 sequence shown in SEQ ID NO:25 and 26.

[0057] The sequence of BMP-12 shown in SEQ ID NO. 1 includes the entiremature sequence and approximately 190 amino acids of the propeptide. Thecoding sequence of the mature human BMP-12 protein appears to begin atnucleotide #496 or #571 and continues through nucleotide #882 of SEQ IDNO:1. The first cysteine in the seven cysteine structure characteristicof TGF-β proteins begins at nucleotide #577. The last cysteine ends at#879. Thus, it is expected that DNA sequences encoding active BMP-12species will comprise nucleotides #577 to #879 of SEQ ID NO:1.

[0058] It is expected that BMP-12, as expressed by mammalian cells suchas CHO cells, exists as a heterogeneous population of active species ofBMP-12 protein with varying N-termini. It is expected that all activespecies will contain the amino acid sequence beginning with the cysteineresidue at amino acid #3 of SEQ ID NO:2 and continue through at leastthe cysteine residue at amino acid 103 or until the stop codon afteramino acid 104. Other active species contain additional amino acidsequence in the N-terminal direction. As described further herein, theN-termini of active species produced by mammalian cells are expected tobegin after the occurrence of a consensus cleavage site, encoding apeptide sequence Arg-X-X-Arg. Thus, it is expected that DNA sequencesencoding active BMP-12 proteins will have a nucleotide sequencecomprising the nucleotide sequence beginning at any of nucleotides #196,199, 208, 217, 361, 388, 493, 496 or 571 to nucleotide #879 or 882 ofSEQ ID NO:1.

[0059] The N-terminus of one active species of human BMP-12 has beenexperimentally determined by expression in E. coli to be as follows:[M]SRXSRKPLHVDF, wherein X designates an amino acid residue with noclear signal, which is consistent with a cysteine residue at thatlocation. Thus, it appears that the N-terminus of this species of BMP-12is at amino acid #1 of SEQ ID NO:1, and a DNA sequence encoding saidspecies of BMP-12 would start at nucleotide #571 of SEQ ID NO:1. Theapparent molecular weight of this species of human BMP-12 dimer wasdetermined by SDS-PAGE to be approximately 20-22 kd on a Novex 16%tricine gel. The pI of this molecule is approximately 4.9. The humanBMP-12 protein exists as a clear, colorless solution in 0.1%trifluoroacetic acid. The N-terminus of another active species of humanBMP-12 has been experimentally determined by expression in E. coli to be[M]TALA. The pI of this molecule is approximately 7.0. The apparentmolecular weight of this species of human BMP-12 dimer was determined bySDS-PAGE to be approximately 25-27 kd on a Novex 16% tricine gel. Thehuman BMP-12 protein exists as a clear, colorless solution in 0.1%trifluoroacetic acid.

[0060] As described earlier, BMP-12 related proteins are a subset of theBMP/TGF-β/Vg-1 family of proteins, including BMP-12 and VL-1, which canbe defined as tendon/ligament-like tissue inducing proteins encoded byDNA sequences which can be cloned and identified, e.g., using PCR, usingBMP-12 specific primers, such as primers #6 and #7 described below, withreduced stringency conditions. It is preferred that DNA sequences of thepresent invention share at least about 80% homology at the amino acidlevel from amino acids with the DNA encoding amino acids #3 to #103 ofSEQ ID NO:1. For the purposes of the present invention, the term BMP-12related proteins does not include the human MP52 protein. Using thesequence information of SEQ ID NO:1 and SEQ ID NO:3, and the comparisonprovided in FIG. 1 it is within the skill of the art to design primersto the BMP-12 sequence which will allow for the cloning of genesencoding BMP-12 related proteins.

[0061] One example of the BMP-12-related proteins of the presentinvention is VL-1, presently referred to as BMP-13. The sequence of thefull mature BMP-13 sequence and at least a part of the propeptide ofBMP-13 is given in SEQ ID NO:25. Like BMP-12, it is expected thatBMP-13, as expressed by mammalian cells such as CHO cells, exists as aheterogeneous population of active species of BMP-13 protein withvarying N-termini. It is expected that all active species will containthe amino acid sequence beginning with the cysteine residue at aminoacid #19 of SEQ ID NO:26 and continue through at least the cysteineresidue at amino acid 119 or until the stop codon after amino acid 120.Other active species contain additional amino acid sequence in theN-terminal direction. As described further herein, the N-termini ofactive species produced by mammalian cells are expected to begin afterthe occurrence of a consensus cleavage site, encoding a peptide sequenceArg-X-X-Arg. Thus, it is expected that DNA sequences encoding activeBMP-13 proteins will have a nucleotide sequence comprising thenucleotide sequence beginning at any of nucleotides #410, 458, 602, 605or 659, to nucleotide #961 or 964 of SEQ ID NO:25.

[0062] In order to produce the purified tendon/ligament-like tissueinducing proteins useful for the present invention, a method is employedcomprising culturing a host cell transformed with a DNA sequencecomprising a suitable coding sequence, particularly the DNA codingsequence from nucleotide #496, #571 or #577 to #879 or #882 of SEQ IDNO:1; and recovering and purifying from the culture medium a proteinwhich contains the amino acid sequence or a substantially homologoussequence as represented by amino acids #-25, #1 or #3 to #103 or #104 ofSEQ ID NO:2. In another embodiment, the method employed comprisesculturing a host cell transformed with a DNA sequence comprising asuitable coding sequence, particularly the DNA coding sequence fromnucleotide #605 or #659 to #961 or #964 of SEQ ID NO:25; and recoveringand purifying from the culture medium a protein which contains the aminoacid sequence or a substantially homologous sequence as represented byamino acids #1 or #19 to #119 or #120 of SEQ ID NO:26.

[0063] The human MP52 DNA is described in WO93/16099, the disclosure ofwhich is incorporated herein by reference. However, this document doesnot disclose the ability of the protein to form tendon/ligament-liketissue, or its use in compositions for induction of tendon/ligament-liketissue. Human MP52 was originally isolated using RNA from human embryotissue. The human MP52 nucleotide sequence (SEQ ID NO:3) and the encodedamino acid sequences (SEQ ID NO:4) are set forth in the SequenceListings herein. The MP52 protein appears to begin at nucleotide #845 ofSEQ ID NO:3 and continues through nucleotide #1204 of SEQ ID NO:3. Thefirst cysteine of the seven cysteine structure characteristic of TGF-βproteins begins at nucleotide #899. The last cysteine ends at #1201.Other active species of MP52 protein may have additional nucleotides atthe N-terminal direction from nucleotide #845 of SEQ ID NO:3.

[0064] Purified human MP52 proteins of the present invention may beproduced by culturing a host cell transformed with a DNA sequencecomprising the DNA coding sequence of SEQ ID NO:3 from nucleotide #845to #1204, and recovering and purifying from the culture medium a proteinwhich contains the amino acid sequence or a substantially homologoussequence as represented by amino acids #1 to #120 of SEQ ID NO:4. It isalso expected that the amino acid sequence from amino acids #17 or #19to #119 or #120 of SEQ ID NO:4 will retain activity. Thus, the DNAsequence from nucleotides #845, #893 or #899 to #1201 or #1204 areexpected to encode active proteins.

[0065] For expression of the protein in mammalian host cells, the hostcell is transformed with a coding sequence encoding a propeptidesuitable for the secretion of proteins by the host cell is linked inproper reading frame to the coding sequence for the mature protein. Forexample, see U.S. Pat. No. 5,168,050, the disclosure of which is herebyincorporated by reference, in which a DNA encoding a precursor portionof a mammalian protein other than BMP-2 is fused to the DNA encoding amature BMP-2 protein. Thus, the present invention includes chimeric DNAmolecules comprising a DNA sequence encoding a propeptide from a memberof the TGF-β superfamily of proteins, is linked in correct reading frameto a DNA sequence encoding a tendon/ligament-like tissue inducingpolypeptide. The term “chimeric” is used to signify that the propeptideoriginates from a different polypeptide than the encoded maturepolypeptide. Of course, the host cell may be transformed with a DNAsequence coding sequence encoding the native propeptide linked incorrect reading frame to a coding sequence encoding the mature proteinshown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:26. The full sequence ofthe native propeptide may be determined through methods known in the artusing the sequences disclosed in SEQ ID NO:1, SEQ ID NO:3, or SEQ IDNO:25 to design a suitable probe for identifying and isolating theentire clone.

[0066] The present invention also encompasses the novel DNA sequences,free of association with DNA sequences encoding other proteinaceousmaterials, and coding for expression of tendon/ligament-like tissueinducing proteins. These DNA sequences include those depicted in SEQ IDNO:1 in a 5′ to 3′ direction and those sequences which hybridize theretounder stringent hybridization conditions [for example, 0.1×SSC, 0.1% SDSat 65° C.; see, T. Maniatis et al, Molecular Cloning (A LaboratoryManual), Cold Spring Harbor Laboratory (1982), pages 387 to 389] andencode a protein having tendon/ligament-like tissue inducing activity.

[0067] Similarly, DNA sequences which code for proteins coded for by thesequences of SEQ ID NO:1 or SEQ ID NO:25, or proteins which comprise theamino acid sequence of SEQ ID NO:2 or SEQ ID NO:26, but which differ incodon sequence due to the degeneracies of the genetic code or allelicvariations (naturally-occurring base changes in the species populationwhich may or may not result in an amino acid change) also encode thetendon/ligament-like tissue inducing proteins described herein.Variations in the DNA sequences of SEQ ID NO:1 or SEQ ID NO:25 which arecaused by point mutations or by induced modifications (includinginsertion, deletion, and substitution) to enhance the activity,half-life or production of the polypeptides encoded are also encompassedin the invention.

[0068] Another aspect of the present invention provides a novel methodfor producing tendon/ligament-like tissue inducing proteins. The methodof the present invention involves culturing a suitable cell line, whichhas been transformed with a DNA sequence encoding a protein of theinvention, under the control of known regulatory sequences. Thetransformed host cells are cultured and the proteins recovered andpurified from the culture medium. The purified proteins aresubstantially free from other proteins with which they are co-producedas well as from other contaminants.

[0069] Suitable cells or cell lines may be mammalian cells, such asChinese hamster ovary cells (CHO). As described above, expression ofprotein in mammalian cells requires an appropriate propeptide to assuresecretion of the protein. The selection of suitable mammalian host cellsand methods for transformation, culture, amplification, screening,product production and purification are known in the art. See, e.g.,Gething and Sambrook, Nature, 293:620-625 (1981), or alternatively,Kaufman et al, Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al,U.S. Pat. No. 4,419,446. Another suitable mammalian cell line, which isdescribed in the accompanying examples, is the monkey COS-1 cell line.The mammalian cell CV-1 may also be suitable.

[0070] Bacterial cells may also be suitable hosts. For example, thevarious strains of E. coli (e.g., HB101, MC1061) are well-known as hostcells in the field of biotechnology. Various strains of B. subtilis,Pseudomonas, other bacilli and the like may also be employed in thismethod. For expression of the protein in bacterial cells, DNA encoding apropeptide is not necessary.

[0071] Bacterial expression of mammalian proteins, including members ofthe TGF-β family is known to produce the proteins in a non-glycosylatedform, and in the form of insoluble pellets, known as inclusion bodies.Techniques have been described in the art for solubilizing theseinclusion bodies, denaturing the protein using a chaotropic agent, andrefolding the protein sufficiently correctly to allow for theirproduction in a soluble form. For example, see EP 0433225, thedisclosure of which is hereby incorporated by reference.

[0072] Alternatively, methods have been devised which circumventinclusion body formation, such as expression of gene fusion proteins,wherein the desired protein is expressed as a fusion protein with afusion partner. The fusion protein is later subjected to cleavage toproduce the desired protein. One example of such a gene fusionexpression system for E. coli is based on use of the E. coli thioredoxingene as a fusion partner, LaVallie et al., Bio/Technology, 11:187-193(1993), the disclosure of which is hereby incorporated by reference.

[0073] Many strains of yeast cells known to those skilled in the art mayalso be available as host cells for expression of the polypeptides ofthe present invention. Additionally, where desired, insect cells may beutilized as host cells in the method of the present invention. See, e.g.Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) andreferences cited therein.

[0074] Another aspect of the present invention provides vectors for usein the method of expression of these tendon/ligament-like tissueinducing proteins. Preferably the vectors contain the full novel DNAsequences described above which encode the novel factors of theinvention. Additionally, the vectors contain appropriate expressioncontrol sequences permitting expression of the protein sequences.Alternatively, vectors incorporating modified sequences as describedabove are also embodiments of the present invention. Additionally, thesequence of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:25 could bemanipulated to express a mature protein by deleting propeptide sequencesand replacing them with sequences encoding the complete propeptides ofBMP proteins or members of the TGF-β superfamily. Thus, the presentinvention includes chimeric DNA molecules encoding a propeptide from amember of the TGF-β superfamily linked in correct reading frame to a DNAsequence encoding a protein having the amino acid sequence of SEQ IDNO:2 or SEQ ID NO:4 or SEQ ID NO:26, The vectors may be employed in themethod of transforming cell lines and contain selected regulatorysequences in operative association with the DNA coding sequences of theinvention which are capable of directing the replication and expressionthereof in selected host cells. Regulatory sequences for such vectorsare known to those skilled in the art and may be selected depending uponthe host cells. Such selection is routine and does not form part of thepresent invention.

[0075] A protein of the present invention, which inducestendon/ligament-like tissue or other tissue formation in circumstanceswhere such tissue is not normally formed, has application in the healingof tendon or ligament tears, deformities and other tendon or ligamentdefects in humans and other animals. Such a preparation employing atendon/ligament-like tissue inducing protein may have prophylactic usein preventing damage to tendon or ligament tissue, as well as use in theimproved fixation of tendon or ligament to bone or other tissues, and inrepairing defects to tendon or ligament tissue. De novotendon/ligament-like tissue formation induced by a composition of thepresent invention contributes to the repair of congenital, traumainduced, or other tendon or ligament defects of other origin, and isalso useful in cosmetic plastic surgery for attachment or repair oftendons or ligaments. The compositions of the invention may also beuseful in the treatment of tendinitis, carpal tunnel syndrome and othertendon or ligament defects. The compositions of the present inventioncan also be used in other indications wherein it is desirable to heal orregenerate tendon and/or ligament tissue. Such indications include,without limitation, regeneration or repair of injuries to theperiodontal ligament, such as occurs in tendonitis, and regeneration orrepair of the tendon-to-bone attachment. The compositions of the presentinvention may provide an environment to attract tendon- orligament-forming cells, stimulate growth of tendon- or ligament-formingcells or induce differentiation of progenitors of tendon- orligament-forming cells.

[0076] The BMP-12 related proteins may be recovered from the culturemedium and purified by isolating them from other proteinaceous materialsfrom which they are co-produced and from other contaminants present. Theproteins of the present invention are capable of inducing the formationof tendon/ligament-like tissue. These proteins may be furthercharacterized by the ability to demonstrate tendon/ligament-like tissueformation activity in the rat ectopic implant assay described below. Itis contemplated that these proteins may have ability to induce theformation of other types of tissue, such as ligaments, as well.

[0077] The tendontligament-like tissue inducing proteins provided hereinalso include factors encoded by the sequences similar to those of SEQ IDNO:1 or SEQ ID NO:25, but into which modifications are naturallyprovided (e.g. allelic variations in the nucleotide sequence which mayresult in amino acid changes in the polypeptide) or deliberatelyengineered. For example, synthetic polypeptides may wholly or partiallyduplicate continuous sequences of the amino acid residues of SEQ IDNO:2. These sequences, by virtue of sharing primary, secondary, ortertiary structural and conformational characteristics withtendon/ligament-like tissue growth factor polypeptides of SEQ ID NO:2may possess tendon/ligament-like or other tissue growth factorbiological properties in common therewith. Thus, they may be employed asbiologically active substitutes for naturally-occurringtendon/ligament-like tissue inducing polypeptides in therapeuticcompositions and processes.

[0078] Other specific mutations of the sequences of tendon/ligament-liketissue inducing proteins described herein involve modifications ofglycosylation sites. These modifications may involve O-linked orN-linked glycosylation sites. For instance, the absence of glycosylationor only partial glycosylation results, from amino acid substitution ordeletion at asparagine-linked glycosylation recognition sites. Theasparagine-linked glycosylation recognition sites comprise tripeptidesequences which are specifically recognized by appropriate cellularglycosylation enzymes. These tripeptide sequences may beasparagine-X-threonine, asparagine-X-serine or asparagine-X-cysteine,where X is usually any amino acid except proline. A variety of aminoacid substitutions or deletions at one or both of the first or thirdamino acid positions of a glycosylation recognition site (and/or aminoacid deletion at the second position) results in non-glycosylation atthe modified tripeptide sequence. Additionally, bacterial expression ofprotein will also result in production of a non-glycosylated protein,even if the glycosylation sites are left unmodified

[0079] The compositions of the present invention comprise a purifiedBMP-12 related protein which may be produced by culturing a celltransformed with the DNA sequence of SEQ ID NO:1 or SEQ ID NO:25 andrecovering and purifying protein having the amino acid sequence of SEQID NO:2 or SEQ ID NO:26 from the culture medium. The purified expressedprotein is substantially free from other proteinaceous materials withwhich it is co-produced, as well as from other contaminants. Therecovered purified protein is contemplated to exhibittendon/ligament-like tissue formation activity, and other tissue growthactivity, such as ligament regeneration. The proteins of the inventionmay be further characterized by the ability to demonstratetendon/ligament-like tissue formation activity in the rat assaydescribed below.

[0080] The compositions for inducing tendon/ligament-like tissueformation of the present invention may comprise an effective amount of atendon/ligament-like tissue inducing protein, wherein said proteincomprises the amino acid sequence of SEQ ID NO:2, preferably amino acids#-25, #1 or #3 to #103 or #104 of SEQ ID NO:2; or amino acids #1 or #19to #120 of SEQ ID NO:26; as well as mutants and/or variants of SEQ IDNO:2 or SEQ ID NO:26, which exhibit the ability to form tendon and/orligament like tissue.

[0081] Compositions of the present invention may further compriseadditional proteins, such as additional members of the TGF-β superfamilyof proteins, such as activins. Another aspect of the invention providespharmaceutical compositions containing a therapeutically effectiveamount of a tendon/ligament-inducing protein, such as BMP-12 or VL-1, ina pharmaceutically acceptable vehicle or carrier. These compositions maybe used to induce the formation of tendon/ligament-like tissue or othertissue. It is contemplated that such compositions may also be used fortendon and ligament repair, wound healing and other tissue repair, suchas skin repair. It is further contemplated that proteins of theinvention may increase neuronal survival and therefore be useful intransplantation and treatment of conditions exhibiting a decrease inneuronal survival. Compositions of the invention may further include atleast one other therapeutically useful agent, such as the BMP proteinsBMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, disclosed forinstance in U.S. Pat. Nos. 5,108,922; 5,013,649; 5,116,738; 5,106,748;5,187,076; and 5,141,905; BMP-8, disclosed in PCT publicationWO91/18098; BMP-9, disclosed in PCT publication WO93/00432; and BMP-10or BMP-11, disclosed in co-pending patent applications, Ser. No.08/061,695 and 08/061,464, filed on May 12, 1993. The disclosure of theabove documents are hereby incorporated by reference herein.

[0082] The compositions of the invention may comprise, in addition to atendon/ligament-inducing protein such as BMP-12 or VL-1 (BMP-13), othertherapeutically useful agents including MP52, epidermal growth factor(EGF), fibroblast growth factor (FGF), platelet derived growth factor(PDGF), transforming growth factors (TGF-α and TGF-β ), and fibroblastgrowth factor-4 (FGF-4), parathyroid hormone (PTH), leukemia inhibitoryfactor (LIF/HILDA/DIA), insulin-like growth factors (IGF-I and IGF-II).Portions of these agents may also be used in compositions of the presentinvention. For example, a composition comprising both BMP-2 and BMP-12implanted together gives rise to both bone and tendon/ligament-liketissue. Such a composition may be useful for treating defects of theembryonic joint where tendon, ligaments, and bone form simultaneously atcontiguous anatomical locations, and may be useful for regeneratingtissue at the site of tendon attachment to bone. It is contemplated thatthe compositions of the invention may also be used in wound healing,such as skin healing and related tissue repair. The types of woundsinclude, but are not limited to burns, incisions and ulcers. (See, e.g.PCT Publication WO84/01106 for discussion of wound healing and relatedtissue repair).

[0083] It is expected that the proteins of the invention may act inconcert with or perhaps synergistically with other related proteins andgrowth factors. Further therapeutic methods and compositions of theinvention therefore comprise a therapeutic amount of at least oneprotein of the invention with a therapeutic amount of at least one ofthe BMP proteins described above. Such compositions may compriseseparate molecules of the BMP proteins or heteromolecules comprised ofdifferent BMP moieties. For example, a method and composition of theinvention may comprise a disulfide linked dimer comprising a BMP-12related protein subunit and a subunit from one of the “BMP” proteinsdescribed above. Thus, the present invention includes compositionscomprising a purified BMP-12 related polypeptide which is a heterodimerwherein one subunit comprises the amino acid sequence from amino acid #1to amino acid #104 of SEQ ID NO:2, and one subunit comprises an aminoacid sequence for a bone morphogenetic protein selected from the groupconsisting of BMP-1, BMP-2, BMP-3, BMP4, BMP-5, BMP-6, BMP-7, BMP-8,BMP-9, BMP-10 and BMP-11. A further embodiment may comprise aheterodimer of disulfide bonded tendon/ligament-like tissue inducingmoieties such as BMP-12, VL-1 (BMP-13) or MP52. For example theheterodimer may comprise one subunit comprising an amino acid sequencefrom #1 to #104 of SEQ ID NO:2 and the other subunit may comprise anamino acid sequence from #1 to #120 of SEQ ID NO:4 or #1 to #120 of SEQID NO:26. Further, compositions of the present invention may be combinedwith other agents beneficial to the treatment of the defect, wound, ortissue in question.

[0084] The preparation and formulation of such physiologicallyacceptable protein compositions, having due regard to pH, isotonicity,stability and the like, is within the skill of the art. The therapeuticcompositions are also presently valuable for veterinary applications dueto the lack of species specificity in TGF-β proteins. Particularlydomestic animals and thoroughbred horses in addition to humans aredesired patients for such treatment with the compositions of the presentinvention.

[0085] The therapeutic method includes administering the compositiontopically, systemically, or locally as an injectable and/or implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Further, the composition, may desirably be encapsulated orinjected in a viscous form for delivery to the site of tissue damage.Topical administration may be suitable for wound healing and tissuerepair. Therapeutically useful agents other than the proteins which mayalso optionally be included in the composition as described above, mayalternatively or additionally, be administered simultaneously orsequentially with the composition in the methods of the invention. Inaddition, the compositions of the present invention may be used inconjunction with presently available treatments for tendon/ligamentinjuries, such as suture (e.g., vicryl sutures or surgical gut sutures,Ethicon Inc., Somerville, N.J.) or tendon/ligament allograft orautograft, in order to enhance or accelerate the healing potential ofthe suture or graft. For example, the suture, allograft or autograft maybe soaked in the compositions of the present invention prior toimplantation. It may also be possible to incorporate the protein orcomposition of the invention onto suture materials, for example, byfreeze-drying.

[0086] The compositions may include an appropriate matrix and/orsequestering agent as a carrier. For instance, the matrix may supportthe composition or provide a surface for tendon/ligament-like tissueformation and/or other tissue formation. The matrix may provide slowrelease of the protein and/or the appropriate environment forpresentation thereof. The sequestering agent may be a substance whichaids in ease of administration through injection or other means, or mayslow the migration of protein from the site of application.

[0087] The choice of a carrier material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are nonbiodegradable and chemicallydefined. Preferred matrices include collagen-based materials, includingsponges, such as Helistat® (Integra LifeSciences, Plainsboro, N.J.), orcollagen in an injectable form, as well as sequestering agents, whichmay be biodegradable, for example hyalouronic acid derived.Biodegradable materials, such as cellulose films, or surgical meshes,may also serve as matrices. Such materials could be sutured into aninjury site, or wrapped around the tendon/ligament.

[0088] Another preferred class of carrier are polymeric matrices,including polymers of poly(lactic acid), poly(glycolic acid) andcopolymers of lactic acid and glycolic acid. These matrices may be inthe form of a sponge, or in the form of porous particles, and may alsoinclude a sequestering agent. Suitable polymer matrices are described,for example, in WO93/00050, the disclosure of which is incorporatedherein by reference.

[0089] Preferred families of sequestering agents include blood, fibrinclot and/or cellulosic materials such as alkylcelluloses (includinghydroxyalkylcelluloses) including methylcellulose, ethylcellulose,hydroxyethylcellulose. hydroxypropylcellulose,hydroxypropyl-methylcellulose, and carboxymethylcellulose. the mostpreferred being cationic salts of carboxymethylcellulose (CMC). Otherpreferred sequestering agents include hyaluronic acid, sodium alginate,poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer andpoly(vinyl alcohol). The amount of sequestering agent useful herein is0.5-20 wt %, preferably 1-10 wt % based on total formulation weight,which represents the amount necessary to prevent desorbtion of theprotein from the polymer matrix and to provide appropriate handling ofthe composition, yet not so much that the progenitor cells are preventedfrom infiltrating the matrix, thereby providing the protein theopportunity to assist the activity of the progenitor cells.

[0090] Additional optional components useful in the practice of thesubject application include, e.g. cryogenic protectors such as mannitol,sucrose, lactose, glucose, or glycine (to protect the protein fromdegradation during lyophilization), antimicrobial preservatives such asmethyl and propyl parabens and benzyl alcohol; antioxidants such asEDTA, citrate and BHT (butylated hydroxytoluene); and surfactants suchas poly(sorbates) and poly(oxyethylenes); etc.

[0091] As described above, the compositions of the invention may beemployed in methods for treating a number of tendon defects, such as theregeneration of tendon/ligament-like tissue in areas of tendon orligament damage, to assist in repair of tears of tendon tissue,ligaments, and various other types of tissue defects or wounds. Thesemethods, according to the invention, entail administering to a patientneeding such tendon/ligament-like tissue or other tissue repair, acomposition comprising an effective amount of a tendon/ligament-liketissue inducing protein, such as described in SEQ ID NO:2, SEQ ID NO:4and/or SEQ ID NO:26. These methods may also entail the administration ofa tendon/ligament-like tissue inducing protein in conjunction with atleast one of the BMP proteins described above.

[0092] In another embodiment, the methods may entail administration of aheterodimeric protein in which one of the monomers is atendon/ligament-like tissue inducing polypeptide, such as BMP-12, VL-1(BMP-13) or MP52, and the second monomer is a member of the TGF-βsuperfamily of growth factors. In addition, these methods may alsoinclude the administration of a tendon/ligament-like tissue inducingprotein with other growth factors including EGF, FGF, TGF-α, TGF-β, andIGF.

[0093] Thus, a further aspect of the invention is a therapeutic methodand composition for repairing tendon/ligament-like tissue, for repairingtendon or ligament as well as treating tendinitis and other conditionsrelated to tendon or ligament defects. Such compositions comprise atherapeutically effective amount of one or more tendon/ligament-liketissue inducing proteins, such as BMP-12, a BMP-12 related protein, orMP52, in admixture with a pharmaceutically acceptable vehicle, carrieror-matrix.

[0094] The dosage regimen will be determined by the attending physicianconsidering various factors which modify the action of the composition,e.g., amount of tendon or ligament tissue desired to be formed, the siteof tendon or ligament damage, the condition of the damaged tendon orligament, the size of a wound, type of damaged tissue, the patient'sage, sex, and diet, the severity of any infection, time ofadministration and other clinical factors. The dosage may vary with thetype of matrix used in the reconstitution and the types of additionalproteins in the composition. The addition of other known growth factors,such as IGF-I (insulin like growth factor I), to the final composition,may also affect the dosage.

[0095] Progress can be monitored by periodic assessment oftendon/ligament-like tissue formation, or tendon or ligament growthand/or repair. The progress can be monitored by methods known in theart, for example, X-rays, arthroscopy, histomorphometric determinationsand tetracycline labeling.

[0096] The following examples illustrate practice of the presentinvention in recovering and characterizing human tendon/ligament-liketissue inducing protein and employing them to recover the othertendon/ligament-like tissue inducing proteins, obtaining the humanproteins, expressing the proteins via recombinant techniques, anddemonstration of the ability of the compositions of the presentinvention to form tendon/ligament-like tissue in an in vivo model.Although the examples demonstrate the invention with respect to BMP-12,with minor modifications within the skill of the art, the same resultsare believed to be attainable with MP52 and VL-1.

EXAMPLE 1

[0097] Isolation of DNA

[0098] DNA sequences encoding BMP-12 and BMP-12 related proteins may beisolated by various techniques known to those skilled in the art. Asdescribed below, oligonucleotide primers may be designed on the basis ofamino acid sequences present in other BMP proteins, Vg-1 relatedproteins and other proteins of the TGF-β superfamily. Regions containingamino acid sequences which are highly conserved within the BMP family ofproteins and within other members of the TGF-β superfamily of proteinscan be identified and consensus amino acid sequences of these highlyconserved regions can be constructed based on the similarity of thecorresponding regions of individual BMP/TGF-β/Vg-1 proteins. An exampleof such a consensus amino acid sequence is indicated below.

[0099] Consensus Amino Acid Sequence (1)

[0100] Trp-Gln/Asn-Asp-Trp-Ile-Val/Ile-Ala (SEQ ID NO:16) Where X/Yindicates that either amino acid residue may appear at that position.

[0101] The following oligonucleotide is designed on the basis of theabove identified consensus amino acid sequence (1):

[0102] #1: CGGATCCTGGVANGAYTGGATHRTNGC (SEQ ID NO:17)

[0103] This oligonucleotide sequence is synthesized on an automated DNAsynthesizer. The standard nucleotide symbols in the above identifiedoligonucleotide primer are as follows: A,adenosine; C,cytosine;G,guanine; T,thymine; N,adenosine or cytosine or guanine or thymine;R,adenosine or cytosine; Y,cytosine or thymine; H,adenosine or cytosineor thymine; V,adenosine or cytosine or guanine; D,adenosine or guanineor thymine.

[0104] The first seven nucleotides of oligonucleotide #1 (underlined)contain the recognition sequence for the restriction endonuclease BamHIin order to facilitate the manipulation of a specifically amplified DNAsequence encoding the BMP-12 protein and are thus not derived from theconsensus amino acid sequence (1) presented above.

[0105] A second consensus amino acid sequence is derived from anotherhighly conserved region of BMP/TGF-β/Vg-1 proteins as described below:

[0106] His-Ala-Ile-Val/Leu-Gln-Thr (SEQ ID NO:18)

[0107] The following oligonucleotide is designed on the basis of theabove identified consensus amino acid sequence (2):

[0108] #2: TTTCTAGAARNGTYTGNACDATNGCRTG (SEQ ID NO:19)

[0109] This oligonucleotide sequence is synthesized on an automated DNAsynthesizer. The same nucleotide symbols are used as described above.

[0110] The first seven nucleotides of oligonucleotide #1 (underlined)contain the recognition sequence for the restriction endonuclease XbaIin order to facilitate the manipulation of a specifically amplified DNAsequence encoding the BMP-12 protein and are thus not derived from theconsensus amino acid sequence (2) presented above.

[0111] It is contemplated that the BMP-12 protein of the invention andother BMP/TGF-β/Vg-1 related proteins may contain amino acid sequencessimilar to the consensus amino acid sequences described above and thatthe location of those sequences within a BMP-12 protein or other novelrelated proteins would correspond to the relative locations in theproteins from which they were derived. It is further contemplated thatthis positional information derived from the structure of otherBMP/TGF-β/Vg-1 proteins and the oligonucleotide sequences #1 and #2which have been derived from consensus amino acid sequences (1) and (2),respectively, could be utilized to specifically amplify DNA sequencesencoding the corresponding amino acids of a BMP-12 protein or otherBMP/TGF-β/Vg-1 related proteins.

[0112] Based on the knowledge of the gene structures of BMP/TGF-β/Vg-1proteins it is further contemplated that human genomic DNA can be usedas a template to perform specific amplification reactions which wouldresult in the identification of BMP-12 BMP/TGF-β/Vg-1 (BMP-12 relatedprotein) encoding sequences. Such specific amplification reactions of ahuman genomic DNA template could be initiated with the use ofoligonucleotide primers #1 and #2 described earlier. Oligonucleotides #1and #2 identified above are utilized as primers to allow the specificamplification of a specific nucleotide sequence from human genomic DNA.The amplification reaction is performed as follows:

[0113] Human genomic DNA (source: peripheral blood lymphocytes),provided by Ken Jacobs of Genetics Institute, is sheared by repeatedpassage through a 25 gauge needle, denatured at 100° C. for 5 minutesand then chilled on ice before adding to a reaction mixture containing200 μM each deoxynucleotide triphosphates (dATP, dGTP, dCTP and dTTP),10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.001% gelatin, 1.25units Taq DNA polymerase, 100 pM oligonucleotide #1 and 100 pMoligonucleotide #2. This reaction mixture is incubated at 94° C. for twominutes and then subjected to thermal cycling in the following manner: 1minute at 94° C., 1 minute at 40° C., 1 minute at 72° C for threecycles; then 1 minute at 94° C., 1 minute at 55° C., 1 minute at 72° C.for thirty-seven cycles, followed by a 10 minute incubation at 72° C.

[0114] The DNA which is specifically amplified by this reaction isethanol precipitated, digested with the restriction endonucleases BamHIand XbaI and subjected to agarose gel electrophoresis. A region of thegel, corresponding to the predicted size of the BMP-12 or otherBMP/TGF-β/Vg-1 encoding DNA fragment, is excised and the specificallyamplified DNA fragments contained therein are electroeluted andsubcloned into the plasmid vector pGEM-3 between the XbaI and BamHIsites of the polylinker. DNA sequence analysis of one of the resultingBMP-12 related subclones indicates the specifically amplified DNAsequence product contained therein encodes a portion of the BMP-12protein of the invention.

[0115] The DNA sequence (SEQ ID NO:5) and derived amino acid sequence(SEQ ID NO:6) of this specifically amplified DNA fragment of BMP-12 areshown in the SEQUENCE Listings.

[0116] Nucleotides #1-#26 of SEQ ID NO:5 comprise a portion ofoligonucleotide #1 and nucleotides #103-#128 comprise a portion of thereverse compliment of oligonucleotide #2 utilized to perform thespecific amplification reaction. Due to the function of oligonucleotides#1 and #2 in initiating the amplification reaction, they may notcorrespond exactly to the actual sequence encoding a BMP-12 protein andare therefore not translated in the corresponding amino acid derivation(SEQ ID NO:6).

[0117] DNA sequence analysis of another subclone indicates that thespecifically amplified DNA product contained therein encodes a portionof another BMP/TGF-β/Vg-1 (BMP-12 related) protein of the inventionnamed VL-1.

[0118] The DNA sequence (SEQ ID NO:7) and derived amino acid sequence(SEQ ID NO:8) of this specifically amplified DNA fragment are shown inthe Sequence Listings.

[0119] Nucleotides #1-#26 of SEQ ID NO:7 comprise a portion ofoligonucleotide #1 and nucleotides #103-#128 comprise a portion of thereverse compliment of oligonucleotide #2 utilized to perform thespecific amplification reaction. Due to the function of oligonucleotides#1 and #2 in initiating the amplification reaction, they may notcorrespond exactly to the actual sequence encoding a VL-1 protein of theinvention and are therefore not translated in the corresponding aminoacid derivation (SEQ ID NO:8).

[0120] The following oligonucleotide probe is designed on the basis ofthe specifically amplified BMP-12 human DNA sequence set forth above(SEQ ID NO:5) and synthesized on an automated DNA synthesizer:

[0121] #3: CCACTGCGAGGGCCTTTGCGACTTCCCTTTGCGTTCGCAC (SEQ ID NO:20)

[0122] This oligonucleotide probe is radioactively labeled with ³²P andemployed to screen a human genomic library constructed in the vectorλFIX (Stratagene catalog #944201). 500,000 recombinants of the humangenomic library are plated at a density of approximately 10,000recombinants per plate on 50 plates. Duplicate nitrocellulose replicasof the recombinant bacteriophage plaques and hybridized tooligonucleotide probe #3 in standard hybridization buffer (SHB=5×SSC,0.1% SDS, 5×Denhardt's, 100 μg/ml salmon sperm DNA) at 65° C. overnight.The following day the radioactively labelled oligonucleotide containinghybridization solution is removed an the filters are washed with0.2×SSC, 0.1% SDS at 65° C. A single positively hybridizing recombinantis identified and plaque purified. This plaque purified recombinantbacteriophage clone which hybridizes to the BMP-12 oligonucleotide probe#3 is designated λHuG-48. A bacteriophage plate stock is made andbacteriophage DNA is isolated from the λHuG-48 human genomic clone. Thebacteriophage λHuG-48 has been deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. “ATCC” under theaccession #75625 on Dec. 7, 1993. This deposit meets the requirements ofthe Budapest Treaty of the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure and Regulationsthereunder. The oligonucleotide hybridizing region of this recombinant,λHuG-48, is localized to a 3.2 kb BamHI fragment. This fragment issubcloned into a plasmid vector (pGEM-3) and DNA sequence analysis isperformed. This plasmid subclone is designated PCR1-1#2 and has beendeposited with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. “ATCC” under the accession #69517 on Dec. 7, 1993.This deposit meets the requirements of the Budapest Treaty of theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and Regulations thereunder. The partial DNAsequence (SEQ ID NO:1) and derived amino acid sequence (SEQ ID NO:2) ofthe 3.2 kb DNA insert of the plasmid subclone PCR1-1#2, derived fromclone λHuG-48, are shown in the Sequence Listings.

[0123] It should be noted that nucleotides #639-#714 of SEQ ID NO:1correspond to nucleotides #27-#102 of the specifically amplified BMP-12encoding DNA fragment set forth in SEQ ID NO:5 thus confirming that thehuman genomic bacteriophage clone λHuG-48 and derivative subclonePCR1-1#2 encode at least a portion of the BMP-12 protein of theinvention. The nucleotide sequence of a portion of the 3.2 kb BamHIinsert of the plasmid PCR1-1#2 contains an open reading frame of atleast 882 base pairs, as defined by nucleotides #1-#882 of SEQ ID NO:1.This open reading frame encodes at least 294 amino acids of the humanBMP-12 protein of the invention. The encoded 294 amino acid human BMP-12protein includes the full mature human BMP-12 protein (amino acids#1-#104 of SEQ ID NO:2), as well as the C-terminal portion of thepropeptide region of the primary translation product (amino acid #-190to #-1 of SEQ ID NO:2).

[0124] Additional DNA sequence of the 3.2 kb BamHI insert of the plasmidPCR1-1#2 set forth in SEQ ID NO:33 demonstrates the presence of an 1164bp open reading frame, as defined by nucleotides #138 through #1301 ofSEQ ID NO:33. [NOTE that all the sequence disclosed in SEQ ID NO:1 iscontained within SEQ ID NO:33]. As this sequence is derived from agenomic clone it is difficult to determine the boundary between the 5′extent of coding sequence and the 3′ limit of intervening sequence(intron/non-coding sequence).

[0125] Based on the knowledge of other BMP proteins and other proteinswithin the TGF-β family, it is predicted that the precursor polypeptidewould be cleaved at the multibasic sequence Arg-Arg-Gly-Arg in agreementwith a proposed consensus proteolytic processing sequence ofArg-X-X-Arg. Cleavage of the BMP-12 precursor polypeptide is expected togenerate a 104 amino acid mature peptide beginning with the amino acidSer at position #1 of SEQ ID NO:2. The processing of BMP-12 into themature form is expected to involve dimerization and removal of theN-terminal region in a manner analogous to the processing of the relatedprotein TGF-β [Gentry et al., Molec & Cell. Biol., 8:4162 (1988);Derynck et al. Nature, 316:701 (1985)].

[0126] It is contemplated therefore that the mature active species ofBMP-12 comprises a homodimer of two polypeptide subunits, each subunitcomprising amino acids #1 to #104 of SEQ ID NO:2 with a predictedmolecular weight of approximately 12,000 daltons. Further active speciesare contemplated comprising at least amino acids #3 to #103 of SEQ IDNO:2, thereby including the first and last conserved cysteine residue.As with other members of the TGF-β/BMP family of proteins, thecarboxy-terminal portion of the BMP-12 protein exhibits greater sequenceconservation than the more amino-terminal portion. The percent aminoacid identity of the human BMP-12 protein in the cysteine-richC-terminal domain (amino acids #3-#104) to the corresponding region ofhuman BMP proteins and other proteins within the TGF-β family is asfollows: BMP-2, 55%; BMP-3, 43%; BMP-4, 53%; BMP-5, 49%; BMP-6, 49%;BMP-7, 50%; BMP-8, 57%; BMP-9, 48%; BMP-10, 57%; activin WC (BMP-11),38%; Vg1, 46%; GDF-1, 47%; TGF-β1, 36%; TGF-β2, 36%; TGF-β3, 39%;inhibin β(B), 36%; inhibin β(A), 41%.

[0127] The human BMP-12 DNA sequence (SEQ ID NO:1), or a portionthereof, can be used as a probe to identify a human cell line or tissuewhich synthesizes BMP-12 mRNA. Briefly described, RNA is extracted froma selected cell or tissue source and either electrophoresed on aformaldehyde agarose gel and transferred to nitrocellulose, or reactedwith formaldehyde and spotted on nitrocellulose directly. Thenitrocellulose is then hybridized to a probe derived from the codingsequence of human BMP-12.

[0128] Alternatively, the human BMP-12 sequence is used to designoligonucleotide primers which will specifically amplify a portion of theBMP-12 encoding sequence located in the region between the primersutilized to perform the specific amplification reaction. It iscontemplated that these human BMP-12 derived primers would allow one tospecifically amplify corresponding BMP-12 encoding sequences from mRNA,cDNA or genomic DNA templates. Once a positive source has beenidentified by one of the above described methods, mRNA is selected byoligo (dT) cellulose chromatography and cDNA is synthesized and clonedin λgt10 or other λ bacteriophage vectors known to those skilled in theart, for example, λZAP by established techniques (Toole et al., supra).It is also possible to perform the oligonucleotide primer directedamplification reaction, described above, directly on a pre-establishedhuman cDNA or genomic library which has been cloned into a λbacteriophage vector. In such cases, a library which yields aspecifically amplified DNA product encoding a portion of the humanBMP-12 protein could be screened directly, utilizing the fragment ofamplified BMP-12 encoding DNA as a probe.

[0129] Oligonucleotide primers designed on the basis of the DNA sequenceof the human BMP-12 genomic clone λHuG-48 are predicted to allow thespecific amplification of human BMP-12 encoding DNA sequences frompre-established human cDNA libraries which are commercially available(ie. Stratagene, La Jolla, Calif. or Clontech Laboratories, Inc., PaloAlto, Calif.). The following oligonucleotide primer is designed on thebasis of nucleotides #571 to #590 of the DNA sequence set forth in SEQID NO:1 and synthesized on an automated DNA synthesizer:

[0130] #4: TGCGGATCCAGCCGCTGCAGCCGCAAGCC (SEQ ID NO:21)

[0131] The first nine nucleotides of primer #4 (underlined) comprise therecognition sequence for the restriction endonuclease BamHI which can beused to facilitate the manipulation of a specifically amplified DNAsequence encoding the human BMP-12 protein of the invention and are thusnot derived from the DNA sequence presented in SEQ ID NO:1. Thefollowing oligonucleotide primer is designed on the basis of nucleotides#866-#885 of the DNA sequence set forth in SEQ ID NO:1 and synthesizedon an automated DNA synthesizer:

[0132] #5 GACTCTAGACTACCTGCAGCCGCAGGCCT (SEQ ID NO:22)

[0133] The first nine nucleotides of primer #5 (underlined) comprise therecognition sequence- for the restriction endonuclease XbaI which can beused to facilitate the manipulation of a specifically amplified DNAsequence encoding the human BMP-12 protein of the invention and are thusnot derived from the DNA sequence presented in SEQ ID NO:1.

[0134] The standard nucleotide symbols in the above identified primersare as follows: A, adenine; C, cytosine; G, guanine; T, thymine.

[0135] Primers #4 and #5 identified above are utilized as primers toallow the amplification of a specific BMP-12 encoding nucleotidesequence from pre-established cDNA libraries which may include thefollowing: human fetal brain cDNA/λZAPII (Stratagene catalog #936206),human liver/λUNI-ZAP XR (Stratagene Catalog #937200), humanlung/λUNI-ZAP XR (Stratagene catalog #937206), and human fetalspleen/UNI-ZAP XR (Stratagene catalog #937205).

[0136] Approximately 1×10⁸ pfu (plaque forming units) of λbacteriophagelibraries containing human cDNA inserts such as those detailed above aredenatured at 95° C. for five minutes prior to addition to a reactionmixture containing 200 μM each deoxynucleotide triphosphates (dATP,dGTP, dCTP and dTTP) 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂,0.001% gelatin, 1.25 units Taq DNA polymerase, 100 pM oligonucleotideprimer #4 and 100 pM oligonucleotide primer #5. The reaction mixture isthen subjected to thermal cycling in the following manner: 1 minute at94° C., 1 minute at 50° C., 1 minute at 72° C. for thirty-nine cyclesfollowed by 10 minutes at 72° C.

[0137] The DNA which is specifically amplified by this reaction would beexpected to generate a BMP-12 encoding product of approximately 333 basepairs, the internal 315 bp of which correspond to nucleotides #571 to#885 of SEQ ID NO:1 and also including 9 bp at each end of the BMP-12specific fragment which correspond to the restriction sites defined bynucleotides #1-#9 of primers #4 and #5. The resulting 333 bp DNA productis digested with the restriction endonucleases BamHI and XbaI, phenolextracted, chloroform extracted and ethanol precipitated.

[0138] Alternatively, to ethanol precipitation, buffer exchange andremoval of small fragments of DNA resulting from the BamHI/XbaIrestriction digest is accomplished by dilution of the digested DNAproduct in 10 mM Tris-HCl pH 8.0, 1 mM EDTA followed by centrifugationthrough a Centricon™ 30 microconcentrator (W.R. Grace & Co., Beverly,Mass.; Product #4209). The resulting BamHI/XbaI digested amplified DNAproduct is subcloned into a plasmid vector (ie. pBluescript, pGEM-3etc.) between the BamHI and XbaI sites of the polylinker region. DNAsequence analysis of the resulting subclones would be required toconfirm the integrity of the BMP-12 encoding insert. Once a positivecDNA source has been identified in this manner, the corresponding cDNAlibrary from which a 333 bp BMP-12 specific sequence was amplified couldbe screened directly with the 333 bp insert or other BMP-12 specificprobes in order to identify and isolate cDNA clones encoding thefull-length BMP-12 protein of the invention.

[0139] Additional methods known to those skilled in the art may be usedto isolate other full-length cDNAs encoding human BMP-12 relatedproteins, or full length cDNA clones encoding BMP-12 related proteins ofthe invention from species other than humans, particularly othermammalian species.

[0140] The following examples demonstrate the use of the human BMP-12sequence to isolate homologues from BMP-12 related proteins in a murinegenomic DNA library.

[0141] The DNA sequence which encodes the human BMP-12 protein of theinvention is predicted to be significantly homologous to BMP-12 andBMP-12 related sequences from species other than humans that it could beutilized to specifically amplify DNA sequences from those other specieswhich would encode the corresponding BMP-12 related proteins.Specifically, the following oligonucleotides are designed on the basisof the human BMP-12 sequence (SEQ ID NO:1) and are synthesized on anautomated DNA synthesizer: #6: GCGGATCCAAGGAGCTCGGCTGGGACGA (SEQ IDNO:23) #7: GGAATTCCCCACCACCATGTCCTCGTAT (SEQ ID NO:24)

[0142] The first eight nucleotides of oligonucleotide primers #6 and #7(underlined) comprise the recognition sequence for the restrictionendonucleases BamHI and EcoRI, respectively. These sequences areutilized to facilitate the manipulation of a specifically amplified DNAsequence encoding a BMP-12 or BMP-12 related protein from a speciesother than human and are thus not derived from the DNA sequencepresented in SEQ ID NO:1. Oligonucleotide primer #6 is designed on thebasis of nucleotides #607-#626 of SEQ ID NO:1 Oligonucleotide primer #7is designed on the basis of the reverse compliment of nucleotides#846-#865 of the DNA sequence set forth in SEQ ID NO:1.

[0143] Oligonucleotide primers #6 and #7 identified above are utilizedas primers to allow the amplification of specific BMP-12 relatedsequences from genomic DNA derived from species other than humans. Theamplification reaction is performed as follows:

[0144] Murine genomic DNA (source: strain Balb c) is sheared by repeatedpassage through a 25 gauge needle, denatured at 100° C. for five minutesand then chilled on ice before adding to a reaction mixture containing200 μM each deoxynucleotide triphosphates (dATP, DGTP, dCTP and dTTP) 10mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.001% gelatin, 1.25 unitsTaq DNA polymerase, 100 pM oligonucleotide primer #6 and 100 pMoligonucleotide primer #7. The reaction mixture is then subjected tothermal cycling in the following manner: 1 minute at 95° C., 1 minute at55° C., 1 minute at 72° C. for forty cycles followed by 10 minutes at72° C.

[0145] The DNA which is specifically amplified by this reaction isethanol precipitated, digested with the restriction endonucleases BamHIand EcoRI and subjected to agarose gel electrophoresis. A region of thegel, corresponding to the predicted size of the murine BMP-12 or BMP-12related encoding DNA fragment, is excised and the specifically amplifiedDNA fragments contained therein are extracted (by electroelution or byother methods known to those skilled in the art) and subcloned in to aplasmid vector, such as pGEM-3 or pBluescript between the BamHI andEcoRI sites of the polylinker. DNA sequence analysis of one of theresulting subclones named mV1, indicates that the specifically amplifiedDNA sequence contained therein encodes a portion of a protein whichappears to be the murine homolog to either the BMP-12 or VL-1 sequenceof the invention. The DNA sequence (SEQ ID NO:10) and derived amino acidsequence (SEQ ID NO:1) of this specifically amplified murine DNAfragment are shown in the sequence listings.

[0146] Nucleotides #1-#26 of SEQ ID NO:10 comprise a portion ofoligonucleotide #6 and nucleotides #246-#272 comprise a portion of thereverse compliment of oligonucleotide #7 utilized to perform thespecific amplification reaction. Nucleotide #27 of SEQ ID NO:10 appearsto be the last nucleotide of a codon triplet, and nucleotides #244-#245of SEQ ID NO:10 appear to be the first two nucleotides of a codontriplet. Therefore, nucleotides #28 to #243 of SEQ ID NO:10 correspondto a partial coding sequence of mV1. Due to the function ofoligonucleotides #6 and #7 in initiating the amplification reaction,they may not correspond exactly to the actual sequence encoding themurine homolog to the human BMP-12 or VL-1 protein of the invention andare therefore not translated in the corresponding amino acid sequencederivation (SEQ ID NO:11).

[0147] Oligonucleotide probes designed on the basis of the specificallyamplified murine BMP-12 or VL-1 DNA sequence set forth in SEQ ID NO:10can be utilized by those skilled in the art to identify full-lengthmurine BMP-12 or VL-1 encoding clones (either cDNA or genomic).

[0148] DNA sequence analysis of another of the resulting subclones namedmV2, indicates that the specifically amplified DNA sequence containedtherein encodes a portion of a murine BMP-12 related sequence of theinvention. The DNA sequence (SEQ ID NO:12) and derived amino acidsequence (SEQ ID NO:13) of this specifically amplified murine DNAfragment are shown in the sequence listings.

[0149] Nucleotides #1-#26 of SEQ ID NO:12 comprise a portion ofoligonucleotide #6 and nucleotides #246-#272 comprise a portion of thereverse compliment of oligonucleotide #7 utilized to perform thespecific amplification reaction. Nucleotide #27 of SEQ ID NO:12 appearsto be the last nucleotide of a codon triplet, and nucleotides #244-#245of SEQ ID NO:12 appear to be the first two nucleotides of a codontriplet. Therefore, nucleotides #28 to #243 of SEQ ID NO:12 correspondto a partial coding sequence of mV2. Due to the function ofoligonucleotides #6 and #7 in initiating the amplification reaction,they may not correspond exactly to the actual sequence encoding themurine BMP-12 related protein of the invention and are therefore nottranslated in the corresponding amino acid sequence derivation. (SEQ IDNO:13).

[0150] Oligonucleotide probes designed on the basis of the specificallyamplified murine BMP-12 related DNA sequence set forth in SEQ ID NO:12can be utilized by those skilled in the art to identify full-lengthmurine BMP-12 related encoding clones (either cDNA or genomic).

[0151] DNA sequence analysis of another of the resulting subclones namedmV9, indicates that the specifically amplified DNA sequence containedtherein encodes a portion of a murine BMP-12 related sequence of theinvention. This sequence appears to be the murine homolog to the humanMP52 DNA sequence described at SEQ ID NO:3. The DNA sequence (SEQ IDNO:14) and derived amino acid sequence (SEQ ID NO:15) of thisspecifically amplified murine DNA fragment are shown in the sequencelistings.

[0152] Nucleotides #1-#26 of SEQ ID NO:14 comprise a portion ofoligonucleotide #6 and nucleotides #246-#272 comprise a portion of thereverse compliment of oligonucleotide #7 utilized to perform-thespecific amplification reaction. Nucleotide #27 of SEQ ID NO:14 appearsto be the last nucleotide of a codon triplet, and nucleotides #244-#245of SEQ ID NO:14 appear to be the first two nucleotides of a codontriplet. Therefore, nucleotides #28 to #243 of SEQ ID NO:14 correspondto a partial coding sequence of mV9. Due to the function ofoligonucleotides #6 and #7 in initiating the amplification reaction,they may not correspond exactly to the actual sequence encoding themurine BMP-12 related protein of the invention and are therefore nottranslated in the corresponding amino acid sequence derivation (SEQ IDNO:15).

[0153] Oligonucleotide probes designed on the basis of the specificallyamplified murine BMP-12 related DNA sequence set forth in SEQ ID NO:14can be utilized by those skilled in the art to identify full-lengthmurine BMP-12 related encoding clones (either cDNA or genomic).

[0154] Alternatively, oligonucleotide primers #6 and #7 identified aboveare utilized as primers to allow the specific amplification of a 275base pair DNA probe, the internal 259 bp of which correspond tonucleotides #607 to #865 of SEQ ID NO:1, from the BMP-12 encodingplasmid subclone PCR1-1#2. This 275bp DNA probe was radioactivelylabelled with ³²P and employed to screen a murine genomic libraryconstructed in the vector λ FIX II (Stratagene catalog #946306). 1million recombinants of the murine genomic library are plated at adensity of approximately 20,000 recombinants per plate on 50 plates.Duplicate nitrocellulose replicas of the recombinant bacteriophageplaques are hybridized, under reduced stringency conditions, to thespecifically amplified 333 bp probe in standard hybridization buffer(SHB=5×SSC, 0.1% SDS, 5×Denhardt's, 100 μg/ml salmon sperm DNA) at 60°C. overnight. The following day the radioactively labelledoligonucleotide containing hybridization solution is removed an thefilters are washed, under reduced stringency conditions, with 2×SSC,0.1% SDS at 60° C. Multiple positively hybridizing recombinants areidentified and plaque purified. Fragments of the positively hybridizingmurine genomic recombinant clones are subcloned into standard plasmidvectors (i.e. pGEM-3) and subjected to DNA sequence analysis.

[0155] DNA sequence analysis of one of these subclones named MVR3indicates that it encodes a portion of the mouse gene corresponding tothe PCR product mV1 (murine homolog of the human BMP-12 sequence setforth in SEQ ID NO:1) described above. The partial DNA sequence of thissubclone and corresponding amino acid translation are set forth in SEQID NO:29 and SEQ ID NO:30 respectively.

[0156] DNA sequence analysis of another one of these subclones namedMVR32 indicates that it encodes a portion of the mouse genecorresponding to the PCR product mV2 (murine homolog of the human VL-1sequence set forth in SEQ ID NO:7) described above. The partial DNAsequence of this subclone and corresponding amino acid translation areset forth in SEQ ID NO:31 and SEQ ID NO:32 respectively.

[0157] DNA sequence analysis of another of these subclones named MVR23indicates that it encodes a portion of the mouse gene corresponding tothe PCR product mV9 (murine homolog of the MP-52 sequence set forth inSEQ ID NO:3) described above.

[0158] In a similar manner to that which is described above foridentifying and isolating human genomic clones encoding the BMP-12protein of the invention, oligonucleotide probe(s) corresponding to theVL-1 encoding sequence set forth in SEQ ID NO:7 can be designed andutilized to identify human genomic or cDNA sequences encoding the VL-1(BMP-13) protein. These oligonucleotides would be designed to regionsspecific for VL-1 encoding sequences and would therefore be likely to bederived from regions of the lowest degree of nucleotide sequenceidentity between the specifically amplified VL-1 encoding sequence (SEQID NO:7) and the specifically amplified BMP-12 encoding sequence (SEQ IDNO:5).

[0159] Alternatively, oligonucleotide primers #4 and #5 identified aboveare utilized as primers to allow the specific amplification of a 333base pair DNA probe, the internal 315 bp of which correspond tonucleotides #571 to #885 of SEQ ID NO:1, from the BMP-12 encodingplasmid subclone PCR1-1#2. This 333 bp DNA probe was radioactivelylabelled with ³²P and employed to screen a human genomic libraryconstructed in the vector λDASH II (Stratagene catalog #945203). 1million recombinants of the human genomic library are plated at adensity of approximately 20,000 recombinants per plate on 50 plates.Duplicate nitrocellulose replicas of the recombinant bacteriophageplaques are hybridized, under reduced stringency conditions, to thespecifically amplified 333 bp probe in standard hybridization buffer(SHB=5×SSC, 0.1% SDS, 5×Denhardt's, 100 μg/ml salmon sperm DNA) at 60°C. overnight. The following day the radioactively labelledoligonucleotide containing hybridization solution is removed an thefilters are washed, under reduced stringency conditions, with 2×SSC,0.1% SDS at 60° C. Multiple (approximately 15) positively hybridizingrecombinants are identified and plaque purified.

[0160] In order to distinguish positively hybridizing recombinantsencoding the VL-1 protein of the invention from BMP-12 and otherBMP-12-related encoding recombinants which would be predicted tohybridize positively to the 333 bp DNA probe generated from the BMP-12encoding plasmid PCR1-1#2 utilized in this screening procedure, thefollowing oligonucleotide probe, based on the VL-1 sequence set forth inSEQ ID NO:7, is designed and synthesized on an automated DNAsynthesizer:

[0161] #8: TGTATGCGACTTCCCGC [SEQUENCE ID NO:35]

[0162] An oligonucleotide corresponding to nucleotides #60 to #76 of SEQID NO:7 which contains 5 nucleotide differences to the correspondingregion of the BMP-12 encoding sequence set forth in SEQ ID NO:1(nucleotides #672 to #689) One of the recombinant bacteriophage cloneswhich hybridizes to the VL-1 oligonucleotide probe #8 is designatedλJLDc31. This recombinant bacteriophage clone is plaque purified, abacteriophage plate stock is made and bacteriophage DNA is isolated fromthe λJLDc31 human genomic clone. The bacteriophage λJLDc31 has beendeposited with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. “ATCC” under the accession #75922 on Oct. 20,1994. This deposit meets the requirements of the Budapest Treaty of theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and Regulations thereunder. Theoligonucleotide hybridizing region of this recombinant, λJLDc31, islocalized to a 2.5 kb Eco RI fragment. This fragment is subcloned into aplasmid vector (pGEM-3) and DNA sequence analysis is performed. Thisplasmid subclone is designated pGEMJLDc31/2.5 and has been depositedwith the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md. “ATCC” under the accession #69710 on Oct. 20, 1994. Thisdeposit meets the requirements of the Budapest Treaty of theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and Regulations thereunder.

[0163] The partial DNA sequence (SEQ ID NO:25) and derived amino acidsequence (SEQ ID NO:26) of a portion of the 2.5 kb DNA insert of theplasmid subclone pGEMJLDc31/2.5, derived from clone λJLDc31, are shownin the Sequence Listings.

[0164] The DNA sequence of a portion of the 2.5 kb EcoRI insert of theplasmid pGEMJLDc31/2.5 is set forth in SEQ ID NO:25, contains an 912 bpopen reading frame, as defined by nucleotides #52 through #963 of SEQ IDNO:25. As this sequence is derived from a genomic clone it is difficultto determine the boundary between the 5′ extent of coding sequence andthe 3′ limit of intervening sequence (intron/non-coding sequence). Theentire open reading frame (nucleotides #52 through #963 of SEQ ID NO:25)encodes a portion of the VL-1 protein of the invention of up to 304amino acids.

[0165] Based on the knowledge of other BMP proteins and other proteinswithin the TGF-β family, it is predicted that the precursor polypeptidewould be cleaved at the multibasic sequence Arg-Arg-Arg-Arg in agreementwith a proposed consensus proteolytic processing sequence ofArg-X-X-Arg. Cleavage of the VL-1 precursor polypeptide is expected togenerate a 120 amino acid mature peptide beginning, with the amino acidThr at position #1 of SEQ ID NO:26. The processing of VL-1 into themature form is expected to involve dimerization and removal of theN-terminal region in a manner analogous to the processing of the relatedprotein TGF-β [Gentry et al., Molec & Cell. Biol., 8:4162 (1988);Derynck et al. Nature, 316:701 (1985)].

[0166] It is contemplated therefore that the mature active species ofVL-1 comprises a homodimer of two polypeptide subunits, each subunitcomprising amino acids #1 to #120 of SEQ ID NO:26 with a predictedmolecular weight of approximately 12,000 daltons. Further active speciesare contemplated comprising at least amino acids #19 to #119 or #120 ofSEQ ID NO:26, thereby including the first and last conserved cysteineresidue.

[0167] Using such a method, a clone encoding the mature human VL-1(BMP-13) was obtained. The nucleotide sequence and corresponding aminoacid sequence encoded by this clone are listed in the Sequence Listingsat SEQ ID NO:25 and 26, respectively.

EXAMPLE 2

[0168] Expression of BMP-12

[0169] In order to produce human BMP-12 proteins, the DNA encoding it istransferred into an appropriate expression vector and introduced intomammalian cells or other preferred eukaryotic or prokaryotic hosts byconventional genetic engineering techniques.

[0170] In order to produce the human BMP-12 protein in bacterial cells,the following procedure is employed.

[0171] Expression of BMP-12 in E. coli

[0172] An expression plasmid pALV1-781, for production of BMP-12 in E.coli was constructed which contains the following principal features.Nucleotides 1-2060 contain DNA sequences originating from the plasmidpUC-18 [Norrander et al., Gene 26:101-106 (1983)] including sequencescontaining the gene for β-lactamase which confers resistance to theantibiotic ampicillin in host E. coli strains, and a colE1-derivedorigin of replication. Nucleotides 2061-2221 contain DNA sequences forthe major leftward promotor (pL) of bacteriophage λ [Sanger et al., J.Mol. Biol. 162:729-773 (1982)], including three operator sequences0_(L)1, 0_(L)2 and 0_(L)3. The operators are the binding sites for λcIrepressor protein, intracellular levels of which control the amount oftranscription initiation from pL. Nucleotides 2222-2723 contain a strongribosome binding sequence included on a sequence derived fromnucleotides 35566 to 35472 and 38137 to 38361 from bacteriophage lambdaas described in Sanger et al., J. Mol. Biol. 162:729-773 (1982).Nucleotides 2724-3041 contain a DNA sequence encoding mature BMP-12protein with all 3′ untranslated sequence removed. The BMP-12 DNAsequences introduced into the pALV1-781 expression vector were modifiedat the 5′ end to raise the A+T content without altering the codingcapacity. These changes were made to increase the efficiency oftranslation initiated on the BMP-12 mRNA in E. coli. Nucleotides3042-3058 provide a “Linker” DNA sequence containing restrictionendonuclease sites. Nucleotides 3059-3127 provide a transcriptiontermination sequence based on that of the E. coli asp A gene [Takagi etal., Nucl. Acids Res. 13:2063-2074 (1985)]. Nucleotides 3128-3532 areDNA sequences derived from pUC-18.

[0173] Plasmid pALV1-781 was transformed into the E. coli host strainGI724 (F, lacI^(q), lacp^(L8), ampC::λcI⁺) by the procedure of Dagertand Ehrlich, Gene 6:23 (1979). GI724 (ATCC accession No. 55151) containsa copy of the wild-type λcI repressor gene stably integrated into thechromosome at the ampC locus, where it has been placed under thetranscriptional control of Salmonella typhimurium trp promotor/operatorsequences. In G1724, λCI protein is made only during growth intryptophan-free media, such as minimal media or a minimal mediumsupplemented with casamino acids such as IMC, described above. Additionof tryptophan to a culture of G1724 will repress the Lrp promoter andturn off synthesis of λcI, gradually causing the induction oftranscription from pL promoters if they are present in the cell.

[0174] Transformants were selected on 1.5% w/v agar plates containingIMC medium, which is composed of M9 medium [Miller, “Experiments inMolecular Genetics,” Cold Spring Harbor Laboratory, New York (1972)]containing 1 mM MgSO₄ and supplemented with 0.5% w/v glucose, 0.2% w/vcasamino acids and 100 μg/ml ampicillin. GI724 transformed withpALV1-781 was grown at 37° C. to an A₅₅₀ of 0.5 in IMC medium containing100 μg/ml ampicillin. Tryptophan was then added to a final concentrationof 100 μg/ml and the culture incubated for a further 4 hours. Duringthis time BMP-12 protein accumulates within the “inclusion body”fraction.

[0175] Preparation of Protein Monomer

[0176] 18 g of frozen cells were weighed out and resuspended in 60 ml of100 mM Tris, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], pH8.3. Cells were lysed by 3 passes through a Microfluidizer™ [model #MCF100 T]. The inclusion body pellet was obtained by centrifugation at15,000 g at 4° C. for 20 minutes. The supernatant was decanted, and thepellet was washed with 100 ml of 100 mM Tris, 1.0 M NaCl, 10 mM EDTA, 1mM PMSF, pH 8.3. The suspension was centrifuged again at 15,000 g at 4°C. for 10 minutes, and the supernatant decanted. The pellet was thenwashed with 100 ml of 100 mM Tris, 10 mM EDTA. 1% Triton X-100, 1 mMPMSF, pH 8.3. The suspension was centrifuged again at 15,000 g at 4° C.for 10 minutes, and the supernatant decanted. The pellet was resuspendedwith 50 ml of 20 mM Tris, 1 mM EDTA, 1 mM PMSF, pH 8.3, containing 1%DTT in a glass tissue homogenizer. Monomeric BMP-12 was then solubilizedby acidification to pH 2.5 with glacial acetic acid. The solublefraction was isolated by centrifugation at 15,000 g for 20 minutes at 4°C.

[0177] The supernatant from this centrifugation was collected andchromatographed over a Sephacryl S-100™ size exclusion column (83 cm×2.6cm; ≈440 ml bed) in 20 ml increments. The Sephacryl S-100™ column wasrun with a mobile phase of 1% acetic acid at a flow rate of 1.4 ml/min.Fractions corresponding to BMP-12 monomer were detected by absorbance at280 nm, and using a computer calculated extinction coefficient of18200M⁻¹ cm⁻¹ and molecular weight (11667 daltons). This size exclusioncolumn pooled material was used as starting material for refoldingreactions.

[0178] As an alternative to the above, 1.0 g of cells stored at −80° C.are measured. Solution (3.4 ml 100 mM TRIS, 10 mM EDTA, pH 8.5) isadded. The solution is vortexed until cells are well suspended. 40 μl100 mM PMSF in isopropanol is added. The cells are lysed at 1000 psi ina French pressure cell. The inclusion bodies are centrifuged at 4° C.for 20 minutes in an Eppendorf microfuge to form pellets. Thesupernatants are decanted. To one pellet (out of 4 total) 1.0 mldegassed 8.0 M guanidine hydrochloride, 0.5 M TRIS, 5 mM EDTA, pH 8.5,containing 250 mM DTT is added. The pellet is dissolved and argon isblown over the liquid for 30 seconds. Next the solution is incubated at37° C. for one hour. Insoluble material is pelleted for 2-3 minutes inan Eppendorf microfuge at 23° C. 0.5-1.0 ml of supernatant is injectedonto a Supelco 2 cm guard cartridge (LC-304), and eluted with anacetonitrile gradient in 0.1% TFA from 1-70% over 35 minutes. BMP-12elutes between 29 and 31 minutes. Fractions are pooled and the proteinconcentration determined by adsorbance at 280 nanometers versus 0.% TFA,using the theoretical extinction coefficient based upon the amino acidcontent.

[0179] As a second alternate method to the above, frozen cell pelletsobtained from the E coli transformants as described above are thawed in30 ml of TE8.3(100:10) buffer (100 mM Tris-HCl pH 8.3, 10 mM Na₂EDTA, 1mM PMSF). Cells are lysed by three passes through a Microfluidizer™[model #MCF 100 T]. The initial inclusion body material pellet isdissolved in 8 M guanidine-HCl, TE8.5(100:10) buffer (100 mM Tris-HCl pH8.5, 10 mM Na₂EDTA which contained 100 mM DTT, and incubated at 37° C.for 1 hour. This material is centrifuged at 12,000×g for 15 minutes atroom temperature.

[0180] Refolding of BMP-12 Protein Using CHAPS System

[0181] A sufficient volume of the BMP-12 pool is lyophilized to give 10μg of protein. 5 μl of glass distilled water is added to redissolve theresidue, then 100 μl of refold mix (50 mM Tris, 1.0 M NaCl, 2%3-(3-chlolamido-propyl)dimethylammonio-1-propane-sulfate (CHAPS), 5 mMEDTA, 2 mM glutathione (reduced) 1 mM glutathione (oxidized); at pH ofapproximately 8.5). The solution is gently mixed and stored at 23° C.for 14 days. Dimer formation is assessed by running an aliquot on aNovex 16% tricine gel at 125 volts for 2.5 hours, followed by CoomassieBlue staining and destaining.

[0182] BMP-12 dimer was purified using a C4 analytical RP-HPLC (reversedphase-high performance liquid chromatography) column (Vydac 214TP54)which was equilibrated to 1% B buffer (diluted into A buffer) and wasrun over 35 minutes. during which the protein elutes, using thefollowing gradient (A buffer=0.1% trifluoroacetic acid, B buffer=95%acetonitrile, 0.1% trifluoroacetic acid [TFA]), with a flow rate of 1ml/min: 1-5 minutes 20% B buffer  5-10 minutes 20-30% B buffer 10-30minutes 30-50% B buffer 30-35 minutes 50-100% B buffer

[0183] Protein was monitored by absorbance at 280 nm. Peak BMP-12fractions (eluting between 29 and 31 minutes) were pooled. Purity wasassessed by SDS-PAGE. The concentration was determined by absorbance at280 nm, and using the computer calculated extinction coefficient andmolecular weight as indicated above.

[0184] Expression of BMP-12 in Mammalian Cells

[0185] Another contemplated preferred expression system for biologicallyactive recombinant human BMP-12 is stably transformed mammalian cells.

[0186] One skilled in the art can construct mammalian expression vectorsby employing the sequence of SEQ ID NO:1, or other DNA sequencesencoding BMP-12 proteins or other modified sequences and known vectors,such as pCD [Okayama et al., Mol. Cell Biol., 2:161-170 (1982)], pJL3,pJL4 [Gough et al., EMBO J., 4:645-653 (1985)] and pMT2 CXM.

[0187] The mammalian expression vector pMT2 CXM is a derivative ofp91023(b) (Wong et al., Science 228:810-815, 1985) differing from thelatter in that it contains the ampicillin resistance gene in place ofthe tetracycline resistance gene and further contains a XhoI site forinsertion of cDNA clones. The functional elements of pMT2 CXM have beendescribed (Kaufman, R. J., 1985, Proc. Natl. Acad. Sci. USA 82:689-693)and include the adenovirus VA genes, the SV40 origin of replicationincluding the 72 bp enhancer, the adenovirus major late promoterincluding a 5′ splice site and the majority of the adenovirus tripartiteleader sequence present on adenovirus late mRNAs, a 3′ splice acceptorsite, a DHFR insert, the SV40 early polyadenylation site (SV40), andpBR322 sequences needed for propagation in E. coli.

[0188] Plasmid pMT2 CXM is obtained by EcoRI digestion of pMT2-VWF,which has been deposited with the American Type Culture Collection(ATCC), Rockville, Md. (USA) under accession number ATCC 67122. EcoRIdigestion excises the cDNA insert present in pMT2-VWF, yielding pMT2 inlinear form which can be ligated and used to transform E. coli HB 101 orDH-5 to ampicillin resistance. Plasmid pMT2 DNA can be prepared byconventional methods. pMT2 CXM is then constructed using loopout/inmutagenesis [Morinaga, et al., Biotechnology 84: 636 (1984). Thisremoves bases 1075 to 1145 relative to the Hind III site near the SV40origin of replication and enhancer sequences of pMT2. In addition itinserts a sequence containing the recognition site for the restrictionendonuclease Xho I. A derivative of pMT2CXM, termed pMT23, containsrecognition sites for the restriction endonucleases PstI, Eco RI, SalIand XhoI. Plasmid pMT2 CXM and pMT23 DNA may be prepared by conventionalmethods.

[0189] pEMC2β1 derived from pMT21 may also be suitable in practice ofthe invention. pMT21 is derived from pMT2 which is derived frompMT2-VWF. As described above EcoRI digestion excises the cDNA insertpresent in pMT-VWF, yielding pMT2 in linear form which can be ligatedand used to transform E. coli HB 101 or DH-5 to ampicillin resistance.Plasmid pMT2 DNA can be prepared by conventional methods.

[0190] pMT21 is derived from pMT2 through the following twomodifications. First, 76 bp of the 5′ untranslated region of the DHFRcDNA including a stretch of 19 G residues from G/C tailing for cDNAcloning is deleted. In this process, a XhoI site is inserted to obtainthe following sequence immediately upstream from DHFR. Second, a uniqueClaI site is introduced by digestion with EcoRV and XbaI, treatment withKlenow fragment of DNA polymerase I, and ligation to a ClaI linker(CATCGATG). This deletes a 250 bp segment from the adenovirus associatedRNA (VAI) region but does not interfere with VAI RNA gene expression orfunction. pMT21 is digested with EcoRI and XhoI, and used to derive thevector pEMC2B1.

[0191] A portion of the EMCV leader is obtained from pMT2-ECAT1 [S. K.Jung, et al, J. Virol 63:1651-1660 (1989)] by digestion with Eco RI andPstI, resulting in a 2752 bp fragment. This fragment is digested withTaqI yielding an Eco RI-TaqI fragment of 508 bp which is purified byelectrophoresis on low melting agarose gel. A 68 bp adapter and itscomplementary strand are synthesized with a 5′ TaqI protruding end and a3′ XhoI protruding end which has a sequence which matches the EMC virusleader sequence from nucleotide 763 to 827. It also changes the ATG atposition 10 within the EMC virus leader to an ATT and is followed by aXhoI site. A three way ligation of the pMT21 Eco RI-XhoI fragment, theEMC virus EcoRI-TaqI fragment, and the 68 bp oligonucleotide adapterTaqI-XhoI adapter resulting in the vector pEMC2β1.

[0192] This vector contains the SV40 origin of replication and enhancer,the adenovirus major late promoter, a cDNA copy of the majority of theadenovirus tripartite leader sequence, a small hybrid interveningsequence, an SV40 polyadenylation signal and the adenovirus VA I gene,DHFR and β-lactamase markers and an EMC sequence, in appropriaterelationships to direct the high level expression of the desired cDNA inmammalian cells.

[0193] The construction of vectors may involve modification of theBMP-12 DNA sequences. For instance, BMP-12 cDNA can be modified byremoving the non-coding nucleotides on the 5′ and 3′ ends of the codingregion. The deleted non-coding nucleotides may or may not be replaced byother sequences known to be beneficial for expression. These vectors aretransformed into appropriate host cells for expression of BMP-12proteins. Additionally, the sequence of SEQ ID NO:1 or other sequencesencoding BMP-12 proteins can be manipulated to express BMP-12 protein byisolating the mature-coding sequence-of nucleotides 571 to 882 of SEQ IDNO:1 and adding at the 5′ end sequences encoding the completepropeptides of other BMP proteins.

[0194] For example, one skilled in the art can make a fusion protein inwhich the propeptide of BMP-2 is linked in operable fashion to themature BMP-12 peptide by preparing a DNA vector in which the DNAsequence encoding the BMP-2 propeptide is linked in proper reading frameto the DNA sequence encoding the mature BMP-12 peptide. The DNA sequenceof such a fusion protein is shown in SEQUENCE ID NO:27.

[0195] One skilled in the art can manipulate the sequences of SEQ IDNO:1 by eliminating or replacing the mammalian regulatory sequencesflanking the coding sequence with bacterial sequences to createbacterial vectors for intracellular or extracellular expression bybacterial cells, as described above. As another example, the codingsequences could be further manipulated (e.g. ligated to other knownlinkers or modified by deleting non-coding sequences therefrom oraltering nucleotides therein by other known techniques). The modifiedBMP-12 coding sequence could then be inserted into a known bacterialvector using procedures such as described in T. Taniguchi et al., Proc.Natl Acad. Sci. USA, 77:5230-5,233 (1980). This exemplary bacterialvector could then be transformed into bacterial host cells and a BMP-12protein expressed thereby. For a strategy for producing extracellularexpression of BMP-12 proteins in bacterial cells, see, e.g. Europeanpatent application EPA 177,343.

[0196] Similar manipulations can be performed for the construction of aninsect vector [See, e.g. procedures described in published Europeanpatent application 155,476] for expression in insect cells. A yeastvector could also be constructed employing yeast regulatory sequencesfor intracellular or extracellular expression of the factors of thepresent invention by yeast cells. [See, e.g., procedures described inpublished PCT application WO86/00639 and European patent application EPA123,289].

[0197] A method for producing high levels of a BMP-12 protein of theinvention in mammalian cells may involve the construction of cellscontaining multiple copies of the heterologous BMP-12 gene. Theheterologous gene is linked to an amplifiable marker, e.g. thedihydrofolate reductase (DHFR) gene for which cells containing increasedgene copies can be selected for propagation in increasing concentrationsof methotrexate (MTX) according to the procedures of Kaufman and Sharp,J. Mol. Biol., 159:601-629 (1982). This approach can be employed with anumber of different cell types.

[0198] For example, a plasmid containing a DNA sequence for a BMP-12 ofthe invention in operative association with other plasmid sequencesenabling expression thereof and the DHFR expression plasmid pAdA26SV(A)3[Kaufman and Sharp, Mol. Cell. Biol., 2:1304 (1982)] can beco-introduced into DHFR-deficient CHO cells, DUKX-BII, by variousmethods including calcium phosphate coprecipitation and transfection,electroporation or protoplast fusion. DHFR expressing transformants areselected for growth in alpha media with dialyzed fetal calf serum, andsubsequently selected for amplification by growth in increasingconcentrations of MTX (e.g. sequential steps in 0.02, 0.2, 1.0 and 5 uMMTX) as described in Kaufman et al., Mol Cell Biol., 5:1750 (1983).Transformants are cloned, and biologically active BMP-12 expression ismonitored by the Rosen-modified Sampath-Reddi rat assay described belowin Example 5. BMP-12 expression should increase with increasing levelsof MTX resistance. BMP-12 polypeptides are characterized using standardtechniques known in the art such as pulse labeling with [35S] methionineor cysteine and polyacrylamide gel electrophoresis. Similar procedurescan be followed to produce other related BMP-12 proteins.

EXAMPLE 3

[0199] Preparation of BMP-2 Propeptide/BMP-12 Mature Peptide Fusion

[0200] In order to construct a vector encoding the BMP-2propeptide/BMP-12 mature peptide fusion, the following cloning procedurewas used to fuse the two sequences together.

[0201] First, a DNA restriction enzyme fragment comprising thepropeptide of human BMP-2 protein, comprising nucleotides 1 through 843of SEQ ID NO:27 is cut from pBMP2ΔEMC. pBMP2ΔEMC is a plasmid derivedfrom lambda U20S-39 (ATCC #40345) comprising the entire coding sequencefor human BMP-2 protein with the non-translated 5′ and 3′ sequences ofBMP-2 deleted from the vector. The 5′ restriction enzyme used was Bgl IIand it cuts pBMP2ΔEMC in the vector at nucleotide 979. The 3′restriction enzyme used was Mae II and it cuts pBMP2ΔEMC in the BMP-2propeptide at nucleotide 1925, just short of the carboxy terminus. Theresulting 954 base pair product was then gel isolated and gene cleaned.Second, a DNA restriction enzyme fragment comprising the 5′ portion ofthe human BMP-12 mature peptide DNA sequence, is cut from pPCR1-1#2 V1-1(ATCC #69517). The 5′ restriction enzyme used was Eae I and it cutspPCR1-1#2 V1-1 just 3′ of N-terminus of the human BMP-12 mature peptidesequence. The resulting 259 base pair product was gel isolated and genecleaned. Third, two DNA oligos were designed and synthesized, so thatwhen annealed would form a tiny DNA fragment comprising fusion sequenceof the extreme 3′ end of the human BMP-2 propeptide and the 5′ end ofBMP-12 mature peptide. The DNA fragment has a 5′ Mae II complimentarysticky end which anneals to the 3′ restriction enzyme fragmentcomprising the human BMP-2 propeptide. The annealed oligo DNA fragmenthas a 3′ Eae I complimentary sticky end which anneals to the 5′ of therestriction enzyme fragment comprising the mature peptide of humanBMP-12. The coding strand oligo is named B2/12 and is 13 base pairslong. Next, a DNA fragment encoding the 123 base pairs at the 3′ end ofthe BMP-12 mature peptide fragment was obtained as follows. First, a DNAfragment comprising the propeptide of human BMP-2 protein, comprisingnucleotides 1 through 846 is PCR amplified from pBMP2ΔEMC. The 5′ primer(oligo 655a) anneals just 5′ of the polylinker. The 3′ primer (BMPpro3)anneals to the BMP-2 propeptide 3′ end and introduces a Bgl IIrestriction enzyme site by silent sequence mutations. The resulting PCRproduct was cut with Sal I, which cleaves in the polylinker, and Bgl II.The 850 base pair restriction enzyme fragment (ending in amino acidsequence REKR) was gel isolated and gene cleaned. The BMP-12 maturepeptide was PCR amplified using a 5′ primer (oligo 5-1) encoding the BglII restriction enzyme site by silent sequence mutations, and annealingto the 5′ end of a possible mature cleavage product, beginning withamino acid sequence SRCS. The 3′ primer (V1-13) anneals to the BMP-12mature peptide 3′ end and introduces a Xba I restriction enzyme siteafter the stop codon. The resulting PCR product was cut with Bgl II andXba I. The 321 base pair restriction enzyme-fragment was gel isolatedand gene cleaned.

[0202] The two restriction fragments were three-way ligated into apreviously SalI and XbaI cut vector. The resultant construct wassequenced to check for PCR induced errors and a silent C to T mutationwas observed at base pair 185 in the propeptide. This plasmid wasdesignated pREKRSRC. Then pREKRSRC was cut with BglII and NgoMI, and thevector fragment encompassing the last 123 base pairs of the BMP12 maturesequence was thereby isolated. The three restriction fragments and theannealed oligolinker were four-way ligated to yield pREKR-TAL with theBMP-2 propeptide with the mature cleavage site at the 3′ end fused tothe (TAL) 5′ end of the BMP-12 mature peptide. The coding sequence ofthe resulting ligated vector is shown in SEQ ID NO:27.

EXAMPLE 4

[0203] Biological Activity of Expressed BMP-12

[0204] To measure the biological activity of the expressed BMP-12proteins obtained in Example 2 above, the proteins are recovered fromthe cell culture and purified by isolating the BMP-12 proteins fromother proteinaceous materials with which they are co-produced as well asfrom other contaminants. The purified protein may be assayed inaccordance with the rat assay described below in Example 5.

[0205] Purification is carried out using standard techniques known tothose skilled in the art.

[0206] Protein analysis is conducted using standard techniques such asSDS-PAGE acrylamide [Laemmli, Nature 227:680 (1970)] stained withCoomassie Blue or silver [Oakley, et al. Anal. Biochem. 105:361 (1980)]and by immunoblot [Towbin, et al. Proc. Natl. Acad. Sci. USA 76:4350(1979)].

EXAMPLE 5

[0207] Rosen Modified Sampath-Reddi Assay

[0208] A modified version of the rat ectopic implant assay described inSampath and Reddi, Proc. Natl. Acad. Sci. USA, 80:6591-6595 (1983) isused to evaluate the activity of the BMP-12 proteins. This modifiedassay is herein called the Rosen-modified Sampath-Reddi assay. The assayhas been widely used to evaluate the bone and cartilage-inducingactivity of BMPs. The ethanol precipitation step of the Sampath-Reddiprocedure is replaced by dialyzing (if the composition is a solution) ordiafiltering (if the composition-is a suspension) the fraction to beassayed against water. The solution or suspension is then equilibratedto 0.1% TFA. The resulting solution is added to 20 mg of rat matrix. Amock rat matrix sample not treated with the protein serves as a control.This material is frozen and lyophilized and the resulting powderenclosed in #5 gelatin capsules. The capsules are implantedsubcutaneously in the abdominal thoracic area of 21-49 day old male LongEvans rats. The implants are removed after 10 days. A section of eachimplant is fixed and processed for histological analysis. 1 μmglycolmethacrylate sections are stained with Von Kossa and acid fuschinto score the amount of induced tendon/ligament-like tissue formationpresent in each implant.

[0209] BMP-12 was implanted in the rats in doses of 1, 5, 25 and 50 μgper implant for 10 days. BMP-2 at a dose of 5 μg was included as apositive control. For all doses of BMP-12 tested, no bone or cartilageformation was observed in the implants after ten days. Instead, theimplants were filled with tissue resembling embryonic tendon, which iseasily recognized by the presence of dense bundles of fibroblastsoriented in the same plane and packed tightly together.[Tendon/ligament-like tissue is described, for example, in Ham andCormack, Histology (JB Lippincott Co. (1979), pp. 367-369, thedisclosure of which is hereby incorporated by reference]. These findingswere reproduced in a second set of assays in which tendon/ligament-liketissues was present in all BMP-12 containing implants. In contrast, theBMP-2 implants, as expected, showed cartilage and bone formation, butcontained no tendon/ligament-like tissue.

[0210] The BMP-12 proteins and related proteins of this invention may beassessed for activity on this assay.

EXAMPLE 6

[0211] Using methods in accordance with the above examples, with minormodifications within the skill of the art, human MP52 protein and themurine homologue of BMP-13 protein were expressed and assayed fortendon/ligament-like tissue inducing activity. All proteins showedcomparable results, similar to those described above for human BMP-12.

[0212] The foregoing descriptions detail presently preferred embodimentsof the present invention. Numerous modifications and variations inpractice thereof are expected to occur to those skilled in the-art uponconsideration of these descriptions. Those modifications and variationsare believed to be encompassed within the claims appended hereto. Thedisclosure of all references discussed herein are hereby incorporated byreference.

1 35 926 base pairs nucleic acid single linear DNA (genomic) Homosapiens v1-1 mat_peptide 571..882 CDS 1..882 1 GCG CGT AAT ACG ACT CACTAT AGG GCG AAT TGG GTA CGG GGC CCA GGC 48 Ala Arg Asn Thr Thr His TyrArg Ala Asn Trp Val Arg Gly Pro Gly -190 -185 -180 -175 AGC TGG ACT TCTCCG CCG TTG CTG CTG CTG TCC ACG TGC CCG GGC GCC 96 Ser Trp Thr Ser ProPro Leu Leu Leu Leu Ser Thr Cys Pro Gly Ala -170 -165 -160 GCC CGA GCGCCA CGC CTG CTG TAC TCG CGG GCA GCT GAG CCC CTA GTC 144 Ala Arg Ala ProArg Leu Leu Tyr Ser Arg Ala Ala Glu Pro Leu Val -155 -150 -145 GGT CAGCGC TGG GAG GCG TTC GAC GTG GCG GAC GCC ATG AGG CGC CAC 192 Gly Gln ArgTrp Glu Ala Phe Asp Val Ala Asp Ala Met Arg Arg His -140 -135 -130 CGTCGT GAA CCG CGC CCC CCC CGC GCG TTC TGC CTC TTG CTG CGC GCA 240 Arg ArgGlu Pro Arg Pro Pro Arg Ala Phe Cys Leu Leu Leu Arg Ala -125 -120 -115GTG GCA GGC CCG GTG CCG AGC CCG TTG GCA CTG CGG CGA CTG GGC TTC 288 ValAla Gly Pro Val Pro Ser Pro Leu Ala Leu Arg Arg Leu Gly Phe -110 -105-100 -95 GGC TGG CCG GGC GGA GGG GGC TCT GCG GCA GAG GAG CGC GCG GTG CTA336 Gly Trp Pro Gly Gly Gly Gly Ser Ala Ala Glu Glu Arg Ala Val Leu -90-85 -80 GTC GTC TCC TCC CGC ACG CAG AGG AAA GAG AGC TTA TTC CGG GAG ATC384 Val Val Ser Ser Arg Thr Gln Arg Lys Glu Ser Leu Phe Arg Glu Ile -75-70 -65 CGC GCC CAG GCC CGC GCG CTC GGG GCC GCT CTG GCC TCA GAG CCG CTG432 Arg Ala Gln Ala Arg Ala Leu Gly Ala Ala Leu Ala Ser Glu Pro Leu -60-55 -50 CCC GAC CCA GGA ACC GGC ACC GCG TCG CCA AGG GCA GTC ATT GGC GGC480 Pro Asp Pro Gly Thr Gly Thr Ala Ser Pro Arg Ala Val Ile Gly Gly -45-40 -35 CGC AGA CGG AGG AGG ACG GCG TTG GCC GGG ACG CGG ACA GCG CAG GGC528 Arg Arg Arg Arg Arg Thr Ala Leu Ala Gly Thr Arg Thr Ala Gln Gly -30-25 -20 -15 AGC GGC GGG GGC GCG GGC CGG GGC CAC GGG CGC AGG GGC CGG AGCCGC 576 Ser Gly Gly Gly Ala Gly Arg Gly His Gly Arg Arg Gly Arg Ser Arg-10 -5 1 TGC AGC CGC AAG CCG TTG CAC GTG GAC TTC AAG GAG CTC GGC TGG GAC624 Cys Ser Arg Lys Pro Leu His Val Asp Phe Lys Glu Leu Gly Trp Asp 5 1015 GAC TGG ATC ATC GCG CCG CTG GAC TAC GAG GCG TAC CAC TGC GAG GGC 672Asp Trp Ile Ile Ala Pro Leu Asp Tyr Glu Ala Tyr His Cys Glu Gly 20 25 30CTT TGC GAC TTC CCT TTG CGT TCG CAC CTC GAG CCC ACC AAC CAT GCC 720 LeuCys Asp Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn His Ala 35 40 45 50ATC ATT CAG ACG CTG CTC AAC TCC ATG GCA CCA GAC GCG GCG CCG GCC 768 IleIle Gln Thr Leu Leu Asn Ser Met Ala Pro Asp Ala Ala Pro Ala 55 60 65 TCCTGC TGT GTG CCA GCG CGC CTC AGC CCC ATC AGC ATC CTC TAC ATC 816 Ser CysCys Val Pro Ala Arg Leu Ser Pro Ile Ser Ile Leu Tyr Ile 70 75 80 GAC GCCGCC AAC AAC GTT GTC TAC AAG CAA TAC GAG GAC ATG GTG GTG 864 Asp Ala AlaAsn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met Val Val 85 90 95 GAG GCC TGCGGC TGC AGG TAGCGCGCGG GCCGGGGAGG GGGCAGCCAC 912 Glu Ala Cys Gly Cys Arg100 GCGGCCGAGG ATCC 926 294 amino acids amino acid linear protein 2 AlaArg Asn Thr Thr His Tyr Arg Ala Asn Trp Val Arg Gly Pro Gly -190 -185-180 -175 Ser Trp Thr Ser Pro Pro Leu Leu Leu Leu Ser Thr Cys Pro GlyAla -170 -165 -160 Ala Arg Ala Pro Arg Leu Leu Tyr Ser Arg Ala Ala GluPro Leu Val -155 -150 -145 Gly Gln Arg Trp Glu Ala Phe Asp Val Ala AspAla Met Arg Arg His -140 -135 -130 Arg Arg Glu Pro Arg Pro Pro Arg AlaPhe Cys Leu Leu Leu Arg Ala -125 -120 -115 Val Ala Gly Pro Val Pro SerPro Leu Ala Leu Arg Arg Leu Gly Phe -110 -105 -100 -95 Gly Trp Pro GlyGly Gly Gly Ser Ala Ala Glu Glu Arg Ala Val Leu -90 -85 -80 Val Val SerSer Arg Thr Gln Arg Lys Glu Ser Leu Phe Arg Glu Ile -75 -70 -65 Arg AlaGln Ala Arg Ala Leu Gly Ala Ala Leu Ala Ser Glu Pro Leu -60 -55 -50 ProAsp Pro Gly Thr Gly Thr Ala Ser Pro Arg Ala Val Ile Gly Gly -45 -40 -35Arg Arg Arg Arg Arg Thr Ala Leu Ala Gly Thr Arg Thr Ala Gln Gly -30 -25-20 -15 Ser Gly Gly Gly Ala Gly Arg Gly His Gly Arg Arg Gly Arg Ser Arg-10 -5 1 Cys Ser Arg Lys Pro Leu His Val Asp Phe Lys Glu Leu Gly Trp Asp5 10 15 Asp Trp Ile Ile Ala Pro Leu Asp Tyr Glu Ala Tyr His Cys Glu Gly20 25 30 Leu Cys Asp Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn His Ala35 40 45 50 Ile Ile Gln Thr Leu Leu Asn Ser Met Ala Pro Asp Ala Ala ProAla 55 60 65 Ser Cys Cys Val Pro Ala Arg Leu Ser Pro Ile Ser Ile Leu TyrIle 70 75 80 Asp Ala Ala Asn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met ValVal 85 90 95 Glu Ala Cys Gly Cys Arg 100 1207 base pairs nucleic acidsingle linear DNA (genomic) Homo sapiens MP52 CDS 845..1204 3 ACCGGGCGGCCCTGAACCCA AGCCAGGACA CCCTCCCCAA ACAAGGCAGG CTACAGCCCG 60 GACTGTGACCCCAAAAGGAC AGCTTCCCGG AGGCAAGGCA CCCCCAAAAG CAGGATCTGT 120 CCCCAGCTCCTTCCTGCTGA AGAAGGCCAG GGAGCCCGGG CCCCCACGAG AGCCCAAGGA 180 GCCGTTTCGCCCACCCCCCA TCACACCCCA CGAGTACATG CTCTCGCTGT ACAGGACGCT 240 GTCCGATGCTGACAGAAAGG GAGGCAACAG CAGCGTGAAG TTGGAGGCTG GCCTGGCCAA 300 CACCATCACCAGCTTTATTG ACAAAGGGCA AGATGACCGA GGTCCCGTGG TCAGGAAGCA 360 GAGGTACGTGTTTGACATTA GTGCCCTGGA GAAGGATGGG CTGCTGGGGG CCGAGCTCCG 420 GATCTTGCGGAAGAAGCCCT CGGACACGGC CAAGCCAGCG GCCCCCGGAG GCGGGCGGGC 480 TGCCCAGCTGAAGCTGTCCA GCTGCCCCAG CGGCCGGCAG CCGGCCTCCT TGCTGGATGT 540 GCGCTCCGTGCCAGGCCTGG ACGGATCTGG CTGGGAGGTG TTCGACATCT GGAAGCTCTT 600 CCGAAACTTTAAGAACTCGG CCCAGCTGTG CCTGGAGCTG GAGGCCTGGG AACGGGGCAG 660 GGCCGTGGACCTCCGTGGCC TGGGCTTCGA CCGCGCCGCC CGGCAGGTCC ACGAGAAGGC 720 CCTGTTCCTGGTGTTTGGCC GCACCAAGAA ACGGGACCTG TTCTTTAATG AGATTAAGGC 780 CCGCTCTGGCCAGGACGATA AGACCGTGTA TGAGTACCTG TTCAGCCAGC GGCGAAAACG 840 GCGG GCC CCACTG GCC ACT CGC CAG GGC AAG CGA CCC AGC AAG AAC CTT 889 Ala Pro Leu AlaThr Arg Gln Gly Lys Arg Pro Ser Lys Asn Leu 1 5 10 15 AAG GCT CGC TGCAGT CGG AAG GCA CTG CAT GTC AAC TTC AAG GAC ATG 937 Lys Ala Arg Cys SerArg Lys Ala Leu His Val Asn Phe Lys Asp Met 20 25 30 GGC TGG GAC GAC TGGATC ATC GCA CCC CTT GAG TAC GAG GCT TTC CAC 985 Gly Trp Asp Asp Trp IleIle Ala Pro Leu Glu Tyr Glu Ala Phe His 35 40 45 TGC GAG GGG CTG TGC GAGTTC CCA TTG CGC TCC CAC CTG GAG CCC ACG 1033 Cys Glu Gly Leu Cys Glu PhePro Leu Arg Ser His Leu Glu Pro Thr 50 55 60 AAT CAT GCA GTC ATC CAG ACCCTG ATG AAC TCC ATG GAC CCC GAG TCC 1081 Asn His Ala Val Ile Gln Thr LeuMet Asn Ser Met Asp Pro Glu Ser 65 70 75 ACA CCA CCC ACC TGC TGT GTG CCCACG CGG CTG AGT CCC ATC AGC ATC 1129 Thr Pro Pro Thr Cys Cys Val Pro ThrArg Leu Ser Pro Ile Ser Ile 80 85 90 95 CTC TTC ATT GAC TCT GCC AAC AACGTG GTG TAT AAG CAG TAT GAG GAC 1177 Leu Phe Ile Asp Ser Ala Asn Asn ValVal Tyr Lys Gln Tyr Glu Asp 100 105 110 ATG GTC GTG GAG TCG TGT GGC TGCAGG TAG 1207 Met Val Val Glu Ser Cys Gly Cys Arg 115 120 120 amino acidsamino acid linear protein 4 Ala Pro Leu Ala Thr Arg Gln Gly Lys Arg ProSer Lys Asn Leu Lys 1 5 10 15 Ala Arg Cys Ser Arg Lys Ala Leu His ValAsn Phe Lys Asp Met Gly 20 25 30 Trp Asp Asp Trp Ile Ile Ala Pro Leu GluTyr Glu Ala Phe His Cys 35 40 45 Glu Gly Leu Cys Glu Phe Pro Leu Arg SerHis Leu Glu Pro Thr Asn 50 55 60 His Ala Val Ile Gln Thr Leu Met Asn SerMet Asp Pro Glu Ser Thr 65 70 75 80 Pro Pro Thr Cys Cys Val Pro Thr ArgLeu Ser Pro Ile Ser Ile Leu 85 90 95 Phe Ile Asp Ser Ala Asn Asn Val ValTyr Lys Gln Tyr Glu Asp Met 100 105 110 Val Val Glu Ser Cys Gly Cys Arg115 120 128 base pairs nucleic acid single linear DNA (genomic) HomoSapiens V1-1 fragment CDS 28..102 5 GGATCCTGGA AGGATTGGAT CATTGCG CCGCTG GAC TAC GAG GCG TAC CAC 51 Pro Leu Asp Tyr Glu Ala Tyr His 1 5 TGCGAG GGC CTT TGC GAC TTC CCT TTG CGT TCG CAC CTC GAG CCC ACC 99 Cys GluGly Leu Cys Asp Phe Pro Leu Arg Ser His Leu Glu Pro Thr 10 15 20 AACCACGCTATAG TCCAAACCTT TCTAGA 128 Asn 25 25 amino acids amino acid linearprotein 6 Pro Leu Asp Tyr Glu Ala Tyr His Cys Glu Gly Leu Cys Asp PhePro 1 5 10 15 Leu Arg Ser His Leu Glu Pro Thr Asn 20 25 128 base pairsnucleic acid single linear DNA (genomic) Homo Sapiens VL-1 CDS 28..102 7GGATCCTGGG ATGACTGGAT TATGGCG CCG CTG GAC TAC GAG GCG TAC CAC 51 Pro LeuAsp Tyr Glu Ala Tyr His 1 5 TGC GAG GGT GTA TGC GAC TTC CCG CTG CGC TCGCAC CTG GAG CCC ACC 99 Cys Glu Gly Val Cys Asp Phe Pro Leu Arg Ser HisLeu Glu Pro Thr 10 15 20 AAC CACGCCATGC TACAAACGCT TCTAGA 128 Asn 25 25amino acids amino acid linear protein 8 Pro Leu Asp Tyr Glu Ala Tyr HisCys Glu Gly Val Cys Asp Phe Pro 1 5 10 15 Leu Arg Ser His Leu Glu ProThr Asn 20 25 3585 base pairs nucleic acid single linear DNA (genomic)pALV1-781 9 CTAACTACCC AACTCAAAAA AAAAAAAAAA AAAAACCCCC TCTAACCCCCATTGACGAAA 60 GGGCCTCGTG ATACGCCTAT TTTTATAGGT TAATGTCATG ATAATAATGGTTTCTTAGAC 120 GTCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT ATTTGTTTATTTTTCTAAAT 180 ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA TAAATGCTTCAATAATATTG 240 AAAAAGGAAG AGTATGAGTA TTCAACATTT CCGTGTCGCC CTTATTCCCTTTTTTGCGGC 300 ATTTTGCCTT CCTGTTTTTG CTCACCCAGA AACGCTGGTG AAAGTAAAAGATGCTGAAGA 360 TCAGTTGGGT GCACGAGTGG GTTACATCGA ACTGGATCTC AACAGCGGTAAGATCCTTGA 420 GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT GATGAGCACT TTTAAAGTTCTGCTATGTGG 480 CGCGGTATTA TCCCGTATTG ACGCCGGGCA AGAGCAACTC GGTCGCCGCATACACTATTC 540 TCAGAATGAC TTGGTTGAGT ACTCACCAGT CACAGAAAAG CATCTTACGGATGGCATGAC 600 AGTAAGAGAA TTATGCAGTG CTGCCATAAC CATGAGTGAT AACACTGCGGCCAACTTACT 660 TCTGACAACG ATCGGAGGAC CGAAGGAGCT AACCGCTTTT TTGCACAACATGGGGGATCA 720 TGTAACTCGC CTTGATCGTT GGGAACCGGA GCTGAATGAA GCCATACCAAACGACGAGCG 780 TGACACCACG ATGCCTGTAG CAATGGCAAC AACGTTGCGC AAACTATTAACTGGCGAACT 840 ACTTACTCTA GCTTCCCGGC AACAATTAAT AGACTGGATG GAGGCGGATAAAGTTGCAGG 900 ACCACTTCTG CGCTCGGCCC TTCCGGCTGG CTGGTTTATT GCTGATAAATCTGGAGCCGG 960 TGAGCGTGGG TCTCGCGGTA TCATTGCAGC ACTGGGGCCA GATGGTAAGCCCTCCCGTAT 1020 CGTAGTTATC TACACGACGG GGAGTCAGGC AACTATGGAT GAACGAAATAGACAGATCGC 1080 TGAGATAGGT GCCTCACTGA TTAAGCATTG GTAACTGTCA GACCAAGTTTACTCATATAT 1140 ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG ATCTAGGTGAAGATCCTTTT 1200 TGATAATCTC ATGACCAAAA TCCCTTAACG TGAGTTTTCG TTCCACTGAGCGTCAGACCC 1260 CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT CTGCGCGTAATCTGCTGCTT 1320 GCAAACAAAA AAACCACCGC TACCAGCGGT GGTTTGTTTG CCGGATCAAGAGCTACCAAC 1380 TCTTTTTCCG AAGGTAACTG GCTTCAGCAG AGCGCAGATA CCAAATACTGTCCTTCTAGT 1440 GTAGCCGTAG TTAGGCCACC ACTTCAAGAA CTCTGTAGCA CCGCCTACATACCTCGCTCT 1500 GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG TCGTGTCTTACCGGGTTGGA 1560 CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC TGAACGGGGGGTTCGTGCAC 1620 ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA TACCTACAGCGTGAGCATTG 1680 AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA GGCGGACAGG TATCCGGTAAGCGGCAGGGT 1740 CGGAACAGGA GAGCGCACGA GGGAGCTTCC AGGGGGAAAC GCCTGGTATCTTTATAGTCC 1800 TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG TGATGCTCGTCAGGGGGGCG 1860 GAGCCTATGG AAAAACGCCA GCAACGCGGC CTTTTTACGG TTCCTGGCCTTTTGCTGGCC 1920 TTTTGCTCAC ATGTTCTTTC CTGCGTTATC CCCTGATTCT GTGGATAACCGTATTACCGC 1980 CTTTGAGTGA GCTGATACCG CTCGCCGCAG CCGAACGACC GAGCGCAGCGAGTCAGTGAG 2040 CGAGGAAGCG GAAGAGCGCC CAATACGCAA ACCGCCTCTC CCCGCGCGTTGGCCGATTCA 2100 TTAATGCAGA ATTGATCTCT CACCTACCAA ACAATGCCCC CCTGCAAAAAATAAATTCAT 2160 ATAAAAAACA TACAGATAAC CATCTGCGGT GATAAATTAT CTCTGGCGGTGTTGACATAA 2220 ATACCACTGG CGGTGATACT GAGCACATCA GCAGGACGCA CTGACCACCATGAAGGTGAC 2280 GCTCTTAAAA ATTAAGCCCT GAAGAAGGGC AGCATTCAAA GCAGAAGGCTTTGGGGTGTG 2340 TGATACGAAA CGAAGCATTG GCCGTAAGTG CGATTCCGGA TTAGCTGCCAATGTGCCAAT 2400 CGCGGGGGGT TTTCGTTCAG GACTACAACT GCCACACACC ACCAAAGCTAACTGACAGGA 2460 GAATCCAGAT GGATGCACAA ACACGCCGCC GCGAACGTCG CGCAGAGAAACAGGCTCAAT 2520 GGAAAGCAGC AAATCCCCTG TTGGTTGGGG TAAGCGCAAA ACCAGTTCCGAAAGATTTTT 2580 TTAACTATAA ACGCTGATGG AAGCGTTTAT GCGGAAGAGG TAAAGCCCTTCCCGAGTAAC 2640 AAAAAAACAA CAGCATAAAT AACCCCGCTC TTACACATTC CAGCCCTGAAAAAGGGCATC 2700 AAATTAAACC ACACCTATGG TGTATGCATT TATTTGCATA CATTCAATCAATTGTTATCT 2760 AAGGAAATAC TTACATATGT CTCGTTGTTC TCGTAAACCA CTGCATGTAGATTTTAAAGA 2820 GCTCGGCTGG GACGACTGGA TCATCGCGCC GCTGGACTAC GAGGCGTACCACTGCGAGGG 2880 CCTTTGCGAC TTCCCTTTGC GTTCGCACCT CGAGCCCACC AACCATGCCATCATTCAGAC 2940 GCTGCTCAAC TCCATGGCAC CAGACGCGGC GCCGGCCTCC TGCTGTGTGCCAGCGCGCCT 3000 CAGCCCCATC AGCATCCTCT ACATCGACGC CGCCAACAAC GTTGTCTACAAGCAATACGA 3060 GGACATGGTG GTGGAGGCCT GCGGCTGCAG GTAGTCTAGA GTCGACCTGCAGTAATCGTA 3120 CAGGGTAGTA CAAATAAAAA AGGCACGTCA GATGACGTGC CTTTTTTCTTGTGAGCAGTA 3180 AGCTTGGCAC TGGCCGTCGT TTTACAACGT CGTGACTGGG AAAACCCTGGCGTTACCCAA 3240 CTTAATCGCC TTGCAGCACA TCCCCCTTTC GCCAGCTGGC GTAATAGCGAAGAGGCCCGC 3300 ACCGATCGCC CTTCCCAACA GTTGCGCAGC CTGAATGGCG AATGGCGCCTGATGCGGTAT 3360 TTTCTCCTTA CGCATCTGTG CGGTATTTCA CACCGCATAT ATGGTGCACTCTCAGTACAA 3420 TCTGCTCTGA TGCCGCATAG TTAAGCCAGC CCCGACACCC GCCAACACCCGCTGACGCGC 3480 CCTGACGGGC TTGTCTGCTC CCGGCATCCG CTTACAGACA AGCTGTGACCGTCTCCGGGA 3540 GCTGCATGTG TCAGAGGTTT TCACCGTCAT CACCGAAACG CGCGA 3585272 base pairs nucleic acid single linear DNA (genomic) mouse mV1 CDS28..243 10 GGATCCAAGG AGCTCGGCTG GGACGAC TGG ATC ATC GCG CCA TTA GAC TAC51 Trp Ile Ile Ala Pro Leu Asp Tyr 1 5 GAG GCA TAC CAC TGC GAG GGC GTTTGC GAC TTT CCT CTG CGC TCG CAC 99 Glu Ala Tyr His Cys Glu Gly Val CysAsp Phe Pro Leu Arg Ser His 10 15 20 CTG GAG CCT ACC AAC CAC GCC ATC ATTCAG ACG CTG CTC AAC TCC ATG 147 Leu Glu Pro Thr Asn His Ala Ile Ile GlnThr Leu Leu Asn Ser Met 25 30 35 40 GCG CCC GAC GCT GCG CCA GCC TCC TGCTGC GTG CCC GCA AGG CTC AGT 195 Ala Pro Asp Ala Ala Pro Ala Ser Cys CysVal Pro Ala Arg Leu Ser 45 50 55 CCC ATC AGC ATT CTC TAC ATC GAT GCC GCCAAC AAC GTG GTC TAC AAG 243 Pro Ile Ser Ile Leu Tyr Ile Asp Ala Ala AsnAsn Val Val Tyr Lys 60 65 70 CAATACGAGG ACATGGTGGT GGGGAATTC 272 72amino acids amino acid linear protein 11 Trp Ile Ile Ala Pro Leu Asp TyrGlu Ala Tyr His Cys Glu Gly Val 1 5 10 15 Cys Asp Phe Pro Leu Arg SerHis Leu Glu Pro Thr Asn His Ala Ile 20 25 30 Ile Gln Thr Leu Leu Asn SerMet Ala Pro Asp Ala Ala Pro Ala Ser 35 40 45 Cys Cys Val Pro Ala Arg LeuSer Pro Ile Ser Ile Leu Tyr Ile Asp 50 55 60 Ala Ala Asn Asn Val Val TyrLys 65 70 272 base pairs nucleic acid single linear DNA (genomic) mousemV2 CDS 28..243 12 GGATCCAAGG AGCTCGGCTG GGACGAC TGG ATT ATC GCG CCC CTAGAG TAC 51 Trp Ile Ile Ala Pro Leu Glu Tyr 1 5 GAG GCC TAT CAC TGC GAGGGC GTG TGC GAC TTT CCG CTG CGC TCG CAC 99 Glu Ala Tyr His Cys Glu GlyVal Cys Asp Phe Pro Leu Arg Ser His 10 15 20 CTT GAG CCC ACT AAC CAT GCCATC ATT CAG ACG CTG ATG AAC TCC ATG 147 Leu Glu Pro Thr Asn His Ala IleIle Gln Thr Leu Met Asn Ser Met 25 30 35 40 GAC CCG GGC TCC ACC CCG CCTAGC TGC TGC GTT CCC ACC AAA CTG ACT 195 Asp Pro Gly Ser Thr Pro Pro SerCys Cys Val Pro Thr Lys Leu Thr 45 50 55 CCC ATT AGC ATC CTG TAC ATC GACGCG GGC AAT AAT GTA GTC TAC AAG 243 Pro Ile Ser Ile Leu Tyr Ile Asp AlaGly Asn Asn Val Val Tyr Lys 60 65 70 CAATACGAGG ACATGGTGGT GGGGAATTC 27272 amino acids amino acid linear protein 13 Trp Ile Ile Ala Pro Leu GluTyr Glu Ala Tyr His Cys Glu Gly Val 1 5 10 15 Cys Asp Phe Pro Leu ArgSer His Leu Glu Pro Thr Asn His Ala Ile 20 25 30 Ile Gln Thr Leu Met AsnSer Met Asp Pro Gly Ser Thr Pro Pro Ser 35 40 45 Cys Cys Val Pro Thr LysLeu Thr Pro Ile Ser Ile Leu Tyr Ile Asp 50 55 60 Ala Gly Asn Asn Val ValTyr Lys 65 70 272 base pairs nucleic acid single linear DNA (genomic)mouse mV9 CDS 28..243 14 GGATCCAAGG AGCTCGGCTG GGACGAC TGG ATC ATC GCACCT CTT GAG TAT 51 Trp Ile Ile Ala Pro Leu Glu Tyr 1 5 GAG GCC TTC CACTGC GAA GGA CTG TGT GAG TTC CCC TTG CGC TCC CAC 99 Glu Ala Phe His CysGlu Gly Leu Cys Glu Phe Pro Leu Arg Ser His 10 15 20 TTG GAG CCC ACA AACCAC GCA GTC ATT CAG ACC CTA ATG AAC TCT ATG 147 Leu Glu Pro Thr Asn HisAla Val Ile Gln Thr Leu Met Asn Ser Met 25 30 35 40 GAC CCT GAA TCC ACACCA CCC ACT TGT TGT GTG CCT ACA CGG CTG AGT 195 Asp Pro Glu Ser Thr ProPro Thr Cys Cys Val Pro Thr Arg Leu Ser 45 50 55 CCT ATT AGC ATC CTC TTCATC GAC TCT GCC AAC AAC GTG GTG TAT AAA 243 Pro Ile Ser Ile Leu Phe IleAsp Ser Ala Asn Asn Val Val Tyr Lys 60 65 70 CAATACGAGG ACATGGTGGTGGGGAATTC 272 72 amino acids amino acid linear protein 15 Trp Ile IleAla Pro Leu Glu Tyr Glu Ala Phe His Cys Glu Gly Leu 1 5 10 15 Cys GluPhe Pro Leu Arg Ser His Leu Glu Pro Thr Asn His Ala Val 20 25 30 Ile GlnThr Leu Met Asn Ser Met Asp Pro Glu Ser Thr Pro Pro Thr 35 40 45 Cys CysVal Pro Thr Arg Leu Ser Pro Ile Ser Ile Leu Phe Ile Asp 50 55 60 Ser AlaAsn Asn Val Val Tyr Lys 65 70 7 amino acids amino acid single linearpeptide BMP/TGF-beta consensus sequence 16 Trp Xaa Asp Trp Ile Xaa Ala 15 27 base pairs nucleic acid single linear DNA (genomic) oligonucleotide#1 17 CGGATCCTGG VANGAYTGGA THRTNGC 27 6 amino acids amino acid singlelinear peptide BMP/TGF-beta consensus sequence 18 His Ala Ile Xaa GlnThr 1 5 28 base pairs nucleic acid single linear DNA (genomic)oligonucleotide #2 19 TTTCTAGAAR NGTYTGNACD ATNGCRTG 28 40 base pairsnucleic acid single linear DNA (genomic) oligonucleotide #3 20CCACTGCGAG GGCCTTTGCG ACTTCCCTTT GCGTTCGCAC 40 29 base pairs nucleicacid single linear DNA (genomic) oligonucleotide #4 21 TGCGGATCCAGCCGCTGCAG CCGCAAGCC 29 29 base pairs nucleic acid single linear DNA(genomic) oligonucleotide #5 22 GACTCTAGAC TACCTGCAGC CGCAGGCCT 29 28base pairs nucleic acid single linear DNA (genomic) oligonucleotide #623 GCGGATCCAA GGAGCTCGGC TGGGACGA 28 28 base pairs nucleic acid singlelinear DNA (genomic) oligonucleotide #7 24 GGAATTCCCC ACCACCATGTCCTCGTAT 28 1171 base pairs nucleic acid single linear DNA (genomic)Human VL-1 protein CDS 2..964 mat_peptide 605..964 25 G AAT TCG GAT CTCTCG CAC ACT CCT CTC CGG AGA CAG AAG TAT TTG 46 Asn Ser Asp Leu Ser HisThr Pro Leu Arg Arg Gln Lys Tyr Leu -201-200 -195 -190 TTT GAT GTG TCCATG CTC TCA GAC AAA GAA GAG CTG GTG GGC GCG GAG 94 Phe Asp Val Ser MetLeu Ser Asp Lys Glu Glu Leu Val Gly Ala Glu -185 -180 -175 CTG CGG CTCTTT CGC CAG GCG CCC TCA GCG CCC TGG GGG CCA CCA GCC 142 Leu Arg Leu PheArg Gln Ala Pro Ser Ala Pro Trp Gly Pro Pro Ala -170 -165 -160 -155 GGGCCG CTC CAC GTG CAG CTC TTC CCT TGC CTT TCG CCC CTA CTG CTG 190 Gly ProLeu His Val Gln Leu Phe Pro Cys Leu Ser Pro Leu Leu Leu -150 -145 -140GAC GCG CGG ACC CTG GAC CCG CAG GGG GCG CCG CCG GCC GGC TGG GAA 238 AspAla Arg Thr Leu Asp Pro Gln Gly Ala Pro Pro Ala Gly Trp Glu -135 -130-125 GTC TTC GAC GTG TGG CAG GGC CTG CGC CAC CAG CCC TGG AAG CAG CTG 286Val Phe Asp Val Trp Gln Gly Leu Arg His Gln Pro Trp Lys Gln Leu -120-115 -110 TGC TTG GAG CTG CGG GCC GCA TGG GGC GAG CTG GAC GCC GGG GAGGCC 334 Cys Leu Glu Leu Arg Ala Ala Trp Gly Glu Leu Asp Ala Gly Glu Ala-105 -100 -95 GAG GCG CGC GCG CGG GGA CCC CAG CAA CCG CCG CCC CCG GACCTG CGG 382 Glu Ala Arg Ala Arg Gly Pro Gln Gln Pro Pro Pro Pro Asp LeuArg -90 -85 -80 -75 AGT CTG GGC TTC GGC CGG AGG GTG CGG CCT CCC CAG GAGCGG GCC CTG 430 Ser Leu Gly Phe Gly Arg Arg Val Arg Pro Pro Gln Glu ArgAla Leu -70 -65 -60 CTG GTG GTA TTC ACC AGA TCC CAG CGC AAG AAC CTG TTCGCA GAG ATG 478 Leu Val Val Phe Thr Arg Ser Gln Arg Lys Asn Leu Phe AlaGlu Met -55 -50 -45 CGC GAG CAG CTG GGC TCG GCC GAG GCT GCG GGC CCG GGCGCG GGC GCC 526 Arg Glu Gln Leu Gly Ser Ala Glu Ala Ala Gly Pro Gly AlaGly Ala -40 -35 -30 GAG GGG TCG TGG CCG CCG CCG TCG GGC GCC CCG GAT GCCAGG CCT TGG 574 Glu Gly Ser Trp Pro Pro Pro Ser Gly Ala Pro Asp Ala ArgPro Trp -25 -20 -15 CTG CCC TCG CCC GGC CGC CGG CGG CGG CGC ACG GCC TTCGCC AGT CGC 622 Leu Pro Ser Pro Gly Arg Arg Arg Arg Arg Thr Ala Phe AlaSer Arg -10 -5 1 5 CAT GGC AAG CGG CAC GGC AAG AAG TCC AGG CTA CGC TGCAGC AAG AAG 670 His Gly Lys Arg His Gly Lys Lys Ser Arg Leu Arg Cys SerLys Lys 10 15 20 CCC CTG CAC GTG AAC TTC AAG GAG CTG GGC TGG GAC GAC TGGATT ATC 718 Pro Leu His Val Asn Phe Lys Glu Leu Gly Trp Asp Asp Trp IleIle 25 30 35 GCG CCC CTG GAG TAC GAG GCC TAT CAC TGC GAG GGT GTA TGC GACTTC 766 Ala Pro Leu Glu Tyr Glu Ala Tyr His Cys Glu Gly Val Cys Asp Phe40 45 50 CCG CTG CGC TCG CAC CTG GAG CCC ACC AAC CAC GCC ATC ATC CAG ACG814 Pro Leu Arg Ser His Leu Glu Pro Thr Asn His Ala Ile Ile Gln Thr 5560 65 70 CTG ATG AAC TCC ATG GAC CCC GGC TCC ACC CCG CCC AGC TGC TGC GTG862 Leu Met Asn Ser Met Asp Pro Gly Ser Thr Pro Pro Ser Cys Cys Val 7580 85 CCC ACC AAA TTG ACT CCC ATC AGC ATT CTA TAC ATC GAC GCG GGC AAT910 Pro Thr Lys Leu Thr Pro Ile Ser Ile Leu Tyr Ile Asp Ala Gly Asn 9095 100 AAT GTG GTC TAC AAG CAG TAC GAG GAC ATG GTG GTG GAG TCG TGC GGC958 Asn Val Val Tyr Lys Gln Tyr Glu Asp Met Val Val Glu Ser Cys Gly 105110 115 TGC AGG TAGCGGTGCC TTTCCCGCCG CCTTGGCCCG GAACCAAGGT GGGCCAAGGT1014 Cys Arg 120 CCGCCTTGCA GGGGAGGCCT GGCTGCAGAG AGGCGGAGGA GGAAGCTGGCGCTGGGGGAG 1074 GCTGAGGGTG AGGGAACAGC CTGGATGTGA GAGCCGGTGG GAGAGAAGGGAGCGCACCTT 1134 CCCAGTAACT TCTACCTGCC AGCCCAGAGG GAAATAT 1171 321 aminoacids amino acid linear protein 26 Asn Ser Asp Leu Ser His Thr Pro LeuArg Arg Gln Lys Tyr Leu Phe -201 -200 -195 -190 Asp Val Ser Met Leu SerAsp Lys Glu Glu Leu Val Gly Ala Glu Leu -185 -180 -175 -170 Arg Leu PheArg Gln Ala Pro Ser Ala Pro Trp Gly Pro Pro Ala Gly -165 -160 -155 ProLeu His Val Gln Leu Phe Pro Cys Leu Ser Pro Leu Leu Leu Asp -150 -145-140 Ala Arg Thr Leu Asp Pro Gln Gly Ala Pro Pro Ala Gly Trp Glu Val-135 -130 -125 Phe Asp Val Trp Gln Gly Leu Arg His Gln Pro Trp Lys GlnLeu Cys -120 -115 -110 Leu Glu Leu Arg Ala Ala Trp Gly Glu Leu Asp AlaGly Glu Ala Glu -105 -100 -95 -90 Ala Arg Ala Arg Gly Pro Gln Gln ProPro Pro Pro Asp Leu Arg Ser -85 -80 -75 Leu Gly Phe Gly Arg Arg Val ArgPro Pro Gln Glu Arg Ala Leu Leu -70 -65 -60 Val Val Phe Thr Arg Ser GlnArg Lys Asn Leu Phe Ala Glu Met Arg -55 -50 -45 Glu Gln Leu Gly Ser AlaGlu Ala Ala Gly Pro Gly Ala Gly Ala Glu -40 -35 -30 Gly Ser Trp Pro ProPro Ser Gly Ala Pro Asp Ala Arg Pro Trp Leu -25 -20 -15 -10 Pro Ser ProGly Arg Arg Arg Arg Arg Thr Ala Phe Ala Ser Arg His -5 1 5 Gly Lys ArgHis Gly Lys Lys Ser Arg Leu Arg Cys Ser Lys Lys Pro 10 15 20 Leu His ValAsn Phe Lys Glu Leu Gly Trp Asp Asp Trp Ile Ile Ala 25 30 35 Pro Leu GluTyr Glu Ala Tyr His Cys Glu Gly Val Cys Asp Phe Pro 40 45 50 55 Leu ArgSer His Leu Glu Pro Thr Asn His Ala Ile Ile Gln Thr Leu 60 65 70 Met AsnSer Met Asp Pro Gly Ser Thr Pro Pro Ser Cys Cys Val Pro 75 80 85 Thr LysLeu Thr Pro Ile Ser Ile Leu Tyr Ile Asp Ala Gly Asn Asn 90 95 100 ValVal Tyr Lys Gln Tyr Glu Asp Met Val Val Glu Ser Cys Gly Cys 105 110 115Arg 120 1233 base pairs nucleic acid single linear DNA (genomic) DNAencoding BMP2 propeptide/BMP-12 mature CDS 1..1233 mat_peptide 847..123327 ATG GTG GCC GGG ACC CGC TGT CTT CTA GCG TTG CTG CTT CCC CAG GTC 48Met Val Ala Gly Thr Arg Cys Leu Leu Ala Leu Leu Leu Pro Gln Val -282-280 -275 -270 CTC CTG GGC GGC GCG GCT GGC CTC GTT CCG GAG CTG GGC CGCAGG AAG 96 Leu Leu Gly Gly Ala Ala Gly Leu Val Pro Glu Leu Gly Arg ArgLys -265 -260 -255 TTC GCG GCG GCG TCG TCG GGC CGC CCC TCA TCC CAG CCCTCT GAC GAG 144 Phe Ala Ala Ala Ser Ser Gly Arg Pro Ser Ser Gln Pro SerAsp Glu -250 -245 -240 -235 GTC CTG AGC GAG TTC GAG TTG CGG CTG CTC AGCATG TTC GGC CTG AAA 192 Val Leu Ser Glu Phe Glu Leu Arg Leu Leu Ser MetPhe Gly Leu Lys -230 -225 -220 CAG AGA CCC ACC CCC AGC AGG GAC GCC GTGGTG CCC CCC TAC ATG CTA 240 Gln Arg Pro Thr Pro Ser Arg Asp Ala Val ValPro Pro Tyr Met Leu -215 -210 -205 GAC CTG TAT CGC AGG CAC TCA GGT CAGCCG GGC TCA CCC GCC CCA GAC 288 Asp Leu Tyr Arg Arg His Ser Gly Gln ProGly Ser Pro Ala Pro Asp -200 -195 -190 CAC CGG TTG GAG AGG GCA GCC AGCCGA GCC AAC ACT GTG CGC AGC TTC 336 His Arg Leu Glu Arg Ala Ala Ser ArgAla Asn Thr Val Arg Ser Phe -185 -180 -175 CAC CAT GAA GAA TCT TTG GAAGAA CTA CCA GAA ACG AGT GGG AAA ACA 384 His His Glu Glu Ser Leu Glu GluLeu Pro Glu Thr Ser Gly Lys Thr -170 -165 -160 -155 ACC CGG AGA TTC TTCTTT AAT TTA AGT TCT ATC CCC ACG GAG GAG TTT 432 Thr Arg Arg Phe Phe PheAsn Leu Ser Ser Ile Pro Thr Glu Glu Phe -150 -145 -140 ATC ACC TCA GCAGAG CTT CAG GTT TTC CGA GAA CAG ATG CAA GAT GCT 480 Ile Thr Ser Ala GluLeu Gln Val Phe Arg Glu Gln Met Gln Asp Ala -135 -130 -125 TTA GGA AACAAT AGC AGT TTC CAT CAC CGA ATT AAT ATT TAT GAA ATC 528 Leu Gly Asn AsnSer Ser Phe His His Arg Ile Asn Ile Tyr Glu Ile -120 -115 -110 ATA AAACCT GCA ACA GCC AAC TCG AAA TTC CCC GTG ACC AGA CTT TTG 576 Ile Lys ProAla Thr Ala Asn Ser Lys Phe Pro Val Thr Arg Leu Leu -105 -100 -95 GACACC AGG TTG GTG AAT CAG AAT GCA AGC AGG TGG GAA AGT TTT GAT 624 Asp ThrArg Leu Val Asn Gln Asn Ala Ser Arg Trp Glu Ser Phe Asp -90 -85 -80 -75GTC ACC CCC GCT GTG ATG CGG TGG ACT GCA CAG GGA CAC GCC AAC CAT 672 ValThr Pro Ala Val Met Arg Trp Thr Ala Gln Gly His Ala Asn His -70 -65 -60GGA TTC GTG GTG GAA GTG GCC CAC TTG GAG GAG AAA CAA GGT GTC TCC 720 GlyPhe Val Val Glu Val Ala His Leu Glu Glu Lys Gln Gly Val Ser -55 -50 -45AAG AGA CAT GTT AGG ATA AGC AGG TCT TTG CAC CAA GAT GAA CAC AGC 768 LysArg His Val Arg Ile Ser Arg Ser Leu His Gln Asp Glu His Ser -40 -35 -30TGG TCA CAG ATA AGG CCA TTG CTA GTA ACT TTT GGC CAT GAT GGA AAA 816 TrpSer Gln Ile Arg Pro Leu Leu Val Thr Phe Gly His Asp Gly Lys -25 -20 -15GGG CAT CCT CTC CAC AAA AGA GAA AAA CGT ACG GCG TTG GCC GGG ACG 864 GlyHis Pro Leu His Lys Arg Glu Lys Arg Thr Ala Leu Ala Gly Thr -10 -5 1 5CGG ACA GCG CAG GGC AGC GGC GGG GGC GCG GGC CGG GGC CAC GGG CGC 912 ArgThr Ala Gln Gly Ser Gly Gly Gly Ala Gly Arg Gly His Gly Arg 10 15 20 AGGGGC CGG AGC CGC TGC AGC CGC AAG CCG TTG CAC GTG GAC TTC AAG 960 Arg GlyArg Ser Arg Cys Ser Arg Lys Pro Leu His Val Asp Phe Lys 25 30 35 GAG CTCGGC TGG GAC GAC TGG ATC ATC GCG CCG CTG GAC TAC GAG GCG 1008 Glu Leu GlyTrp Asp Asp Trp Ile Ile Ala Pro Leu Asp Tyr Glu Ala 40 45 50 TAC CAC TGCGAG GGC CTT TGC GAC TTC CCT TTG CGT TCG CAC CTC GAG 1056 Tyr His Cys GluGly Leu Cys Asp Phe Pro Leu Arg Ser His Leu Glu 55 60 65 70 CCC ACC AACCAT GCC ATC ATT CAG ACG CTG CTC AAC TCC ATG GCA CCA 1104 Pro Thr Asn HisAla Ile Ile Gln Thr Leu Leu Asn Ser Met Ala Pro 75 80 85 GAC GCG GCG CCGGCC TCC TGC TGT GTG CCA GCG CGC CTC AGC CCC ATC 1152 Asp Ala Ala Pro AlaSer Cys Cys Val Pro Ala Arg Leu Ser Pro Ile 90 95 100 AGC ATC CTC TACATC GAC GCC GCC AAC AAC GTT GTC TAC AAG CAA TAC 1200 Ser Ile Leu Tyr IleAsp Ala Ala Asn Asn Val Val Tyr Lys Gln Tyr 105 110 115 GAG GAC ATG GTGGTG GAG GCC TGC GGC TGC AGG 1233 Glu Asp Met Val Val Glu Ala Cys Gly CysArg 120 125 411 amino acids amino acid linear protein 28 Met Val Ala GlyThr Arg Cys Leu Leu Ala Leu Leu Leu Pro Gln Val -282 -280 -275 -270 LeuLeu Gly Gly Ala Ala Gly Leu Val Pro Glu Leu Gly Arg Arg Lys -265 -260-255 Phe Ala Ala Ala Ser Ser Gly Arg Pro Ser Ser Gln Pro Ser Asp Glu-250 -245 -240 -235 Val Leu Ser Glu Phe Glu Leu Arg Leu Leu Ser Met PheGly Leu Lys -230 -225 -220 Gln Arg Pro Thr Pro Ser Arg Asp Ala Val ValPro Pro Tyr Met Leu -215 -210 -205 Asp Leu Tyr Arg Arg His Ser Gly GlnPro Gly Ser Pro Ala Pro Asp -200 -195 -190 His Arg Leu Glu Arg Ala AlaSer Arg Ala Asn Thr Val Arg Ser Phe -185 -180 -175 His His Glu Glu SerLeu Glu Glu Leu Pro Glu Thr Ser Gly Lys Thr -170 -165 -160 -155 Thr ArgArg Phe Phe Phe Asn Leu Ser Ser Ile Pro Thr Glu Glu Phe -150 -145 -140Ile Thr Ser Ala Glu Leu Gln Val Phe Arg Glu Gln Met Gln Asp Ala -135-130 -125 Leu Gly Asn Asn Ser Ser Phe His His Arg Ile Asn Ile Tyr GluIle -120 -115 -110 Ile Lys Pro Ala Thr Ala Asn Ser Lys Phe Pro Val ThrArg Leu Leu -105 -100 -95 Asp Thr Arg Leu Val Asn Gln Asn Ala Ser ArgTrp Glu Ser Phe Asp -90 -85 -80 -75 Val Thr Pro Ala Val Met Arg Trp ThrAla Gln Gly His Ala Asn His -70 -65 -60 Gly Phe Val Val Glu Val Ala HisLeu Glu Glu Lys Gln Gly Val Ser -55 -50 -45 Lys Arg His Val Arg Ile SerArg Ser Leu His Gln Asp Glu His Ser -40 -35 -30 Trp Ser Gln Ile Arg ProLeu Leu Val Thr Phe Gly His Asp Gly Lys -25 -20 -15 Gly His Pro Leu HisLys Arg Glu Lys Arg Thr Ala Leu Ala Gly Thr -10 -5 1 5 Arg Thr Ala GlnGly Ser Gly Gly Gly Ala Gly Arg Gly His Gly Arg 10 15 20 Arg Gly Arg SerArg Cys Ser Arg Lys Pro Leu His Val Asp Phe Lys 25 30 35 Glu Leu Gly TrpAsp Asp Trp Ile Ile Ala Pro Leu Asp Tyr Glu Ala 40 45 50 Tyr His Cys GluGly Leu Cys Asp Phe Pro Leu Arg Ser His Leu Glu 55 60 65 70 Pro Thr AsnHis Ala Ile Ile Gln Thr Leu Leu Asn Ser Met Ala Pro 75 80 85 Asp Ala AlaPro Ala Ser Cys Cys Val Pro Ala Arg Leu Ser Pro Ile 90 95 100 Ser IleLeu Tyr Ile Asp Ala Ala Asn Asn Val Val Tyr Lys Gln Tyr 105 110 115 GluAsp Met Val Val Glu Ala Cys Gly Cys Arg 120 125 1203 base pairs nucleicacid single linear DNA (genomic) murine MV1 CDS 2..721 29 A AAG TTC TGCCTG GTG CTG GNG NCG GTG ACG GCC TCG GAG AGC AGN 46 Lys Phe Cys Leu ValLeu Xaa Xaa Val Thr Ala Ser Glu Ser Xaa 1 5 10 15 CNG CTG GCC CTG AGACGA CTG GGC TTC GGC TGN CCG GGC GGT GGC GAC 94 Xaa Leu Ala Leu Arg ArgLeu Gly Phe Gly Xaa Pro Gly Gly Gly Asp 20 25 30 GGC GGC GGC ACT GCG GNCGAG GAG CGC GCG CTG TTG GTG ATC TCC TCC 142 Gly Gly Gly Thr Ala Xaa GluGlu Arg Ala Leu Leu Val Ile Ser Ser 35 40 45 CGT ACG CAA AGG AAA GAG AGTCTG TTC CGG GAG ATC CGA GCC CAG GCC 190 Arg Thr Gln Arg Lys Glu Ser LeuPhe Arg Glu Ile Arg Ala Gln Ala 50 55 60 CGT GCT CTC CGG GCC GCT GCA GAGCCG CCA CCG GAT CCA GGA CCA GGC 238 Arg Ala Leu Arg Ala Ala Ala Glu ProPro Pro Asp Pro Gly Pro Gly 65 70 75 GCT GGG TCA CGC AAA GCC AAC CTG GGCGGT CGC AGG CGG CAG CGG ACT 286 Ala Gly Ser Arg Lys Ala Asn Leu Gly GlyArg Arg Arg Gln Arg Thr 80 85 90 95 GCG CTG GCT GGG ACT CGG GGA GNG NAGGGA AGC GGT GGT GGC GGC GGT 334 Ala Leu Ala Gly Thr Arg Gly Xaa Xaa GlySer Gly Gly Gly Gly Gly 100 105 110 GGC GGT GGC GGC GGC GGC GGC GGC GGCGGC GGC GGC GGC GGC GGC GCA 382 Gly Gly Gly Gly Gly Gly Gly Gly Gly GlyGly Gly Gly Gly Gly Ala 115 120 125 GGC AGG GGC CAC GGG CGC AGA GGC CGGAGC CGC TGC GGT CGC AAG TCA 430 Gly Arg Gly His Gly Arg Arg Gly Arg SerArg Cys Gly Arg Lys Ser 130 135 140 CTG CAC GTG GAC TTT AAG GAG CTG GGCTGG GAC GAC TGG ATC ATC GCG 478 Leu His Val Asp Phe Lys Glu Leu Gly TrpAsp Asp Trp Ile Ile Ala 145 150 155 CCA TTA GAC TAC GAG GCA TAC CAC TGCGAG GGC GTT TGC GAC TTT CCT 526 Pro Leu Asp Tyr Glu Ala Tyr His Cys GluGly Val Cys Asp Phe Pro 160 165 170 175 CTG CGC TCG CAC CTG GAG CCT ACCAAC CAC GCC ATC ATT CAG ACG CTG 574 Leu Arg Ser His Leu Glu Pro Thr AsnHis Ala Ile Ile Gln Thr Leu 180 185 190 CTC AAC TCC ATG GCG CCC GAC GCTGCG CCA GCC TCC TGC TGC GTG CCC 622 Leu Asn Ser Met Ala Pro Asp Ala AlaPro Ala Ser Cys Cys Val Pro 195 200 205 GCA AGG CTC AGT CCC ATC AGC ATTCTC TAC ATC GAT GCC GCC AAC AAC 670 Ala Arg Leu Ser Pro Ile Ser Ile LeuTyr Ile Asp Ala Ala Asn Asn 210 215 220 GTG GTC TAC AAG CAG TAC GAA GACATG GTG GTG GAG GCC TGC GGC TGC 718 Val Val Tyr Lys Gln Tyr Glu Asp MetVal Val Glu Ala Cys Gly Cys 225 230 235 AGG TAGCATGCGG TCTGGGGAGGGTCTGGCCGC CCAGGACCCT AGCTCAAGAG 771 Arg 240 CAGGTGTCAT CAGGCCCGAGGGACGGCGGA CTATGGCCTC TGCCAGCACA GAGGAGAGCA 831 CACAGTTAAC ACTCACATTTACACACTCCT TCACTCACGC ACATGTTTAC CGTGGACGGC 891 AGGCGCTAAA AGCCTTGCTTATTTGCTACC ATTGATACAA ACCTCTGTCC TTTTCGGGAG 951 AGGGAAGGGC ATCTGTGTTTATGTTGCAGT AATTGGCACT AAATCCAAGT AGAAATGGGT 1011 TAGCATTGGA TTCTCCTTTTAGTTGGAGGC GGTGTGGCTG GATTCCTGAC GTTGGATATG 1071 GAGTGCACTG CAGGGCTGGGATACCCAGAT TCTCTGGAGT GGGCATTGGG AACCTTCAAA 1131 AGTAAGGAGC CACTGGGGCTTGGGAGGGAG CACCCGGTTC CTAAACAAGT CTGATGTGTA 1191 CTGCTCAGTT TG 1203 240amino acids amino acid linear protein 30 Lys Phe Cys Leu Val Leu Xaa XaaVal Thr Ala Ser Glu Ser Xaa Xaa 1 5 10 15 Leu Ala Leu Arg Arg Leu GlyPhe Gly Xaa Pro Gly Gly Gly Asp Gly 20 25 30 Gly Gly Thr Ala Xaa Glu GluArg Ala Leu Leu Val Ile Ser Ser Arg 35 40 45 Thr Gln Arg Lys Glu Ser LeuPhe Arg Glu Ile Arg Ala Gln Ala Arg 50 55 60 Ala Leu Arg Ala Ala Ala GluPro Pro Pro Asp Pro Gly Pro Gly Ala 65 70 75 80 Gly Ser Arg Lys Ala AsnLeu Gly Gly Arg Arg Arg Gln Arg Thr Ala 85 90 95 Leu Ala Gly Thr Arg GlyXaa Xaa Gly Ser Gly Gly Gly Gly Gly Gly 100 105 110 Gly Gly Gly Gly GlyGly Gly Gly Gly Gly Gly Gly Gly Gly Ala Gly 115 120 125 Arg Gly His GlyArg Arg Gly Arg Ser Arg Cys Gly Arg Lys Ser Leu 130 135 140 His Val AspPhe Lys Glu Leu Gly Trp Asp Asp Trp Ile Ile Ala Pro 145 150 155 160 LeuAsp Tyr Glu Ala Tyr His Cys Glu Gly Val Cys Asp Phe Pro Leu 165 170 175Arg Ser His Leu Glu Pro Thr Asn His Ala Ile Ile Gln Thr Leu Leu 180 185190 Asn Ser Met Ala Pro Asp Ala Ala Pro Ala Ser Cys Cys Val Pro Ala 195200 205 Arg Leu Ser Pro Ile Ser Ile Leu Tyr Ile Asp Ala Ala Asn Asn Val210 215 220 Val Tyr Lys Gln Tyr Glu Asp Met Val Val Glu Ala Cys Gly CysArg 225 230 235 240 1046 base pairs nucleic acid single linear DNA(genomic) NO NO MURINE MV2 CDS 2..790 31 A AGA AAA CAA GCT TGC ATT CCTGCA GGT CCG ACT CTA AGA GGA TCC 46 Arg Lys Gln Ala Cys Ile Pro Ala GlyPro Thr Leu Arg Gly Ser 1 5 10 15 TCA GGG ACC CAA CCC AGG CCG GCT GGGAAG TCT TTC GAC GTG TGG CAG 94 Ser Gly Thr Gln Pro Arg Pro Ala Gly LysSer Phe Asp Val Trp Gln 20 25 30 GGC CTG CGC CCT CAG CCT TGG AAG CAG CTGTGC CTG GAG TTG CGG GCA 142 Gly Leu Arg Pro Gln Pro Trp Lys Gln Leu CysLeu Glu Leu Arg Ala 35 40 45 GCC TGG GGT GAG CTG GAC RCC GGG GAT ACG GGGGCG CGC GCG AGG GGT 190 Ala Trp Gly Glu Leu Asp Xaa Gly Asp Thr Gly AlaArg Ala Arg Gly 50 55 60 CCC CAG CAG CCA CCG CCT CTG GAC CTG CGG AGT CTGGGC TTC GGT CGG 238 Pro Gln Gln Pro Pro Pro Leu Asp Leu Arg Ser Leu GlyPhe Gly Arg 65 70 75 AGG GTG AGA CCG CCC CAG GAG CGC GCC CTG CTT GTA GTGTTC ACC AGA 286 Arg Val Arg Pro Pro Gln Glu Arg Ala Leu Leu Val Val PheThr Arg 80 85 90 95 TCG CAG CGC AAG AAC CTG TTC ACT GAG ATG CAT GAG CAGCTG GGC TCT 334 Ser Gln Arg Lys Asn Leu Phe Thr Glu Met His Glu Gln LeuGly Ser 100 105 110 GCA GAG GCT GCG GGA GCC GAG GGG TCA TGT CCA GCG CCGTCG GGC TCC 382 Ala Glu Ala Ala Gly Ala Glu Gly Ser Cys Pro Ala Pro SerGly Ser 115 120 125 CCA GAC ACC GGG TCT TGG CTG CCC TCG CCC GGC CGC CGGCGG CGA CGC 430 Pro Asp Thr Gly Ser Trp Leu Pro Ser Pro Gly Arg Arg ArgArg Arg 130 135 140 ACC GCC TTC GCC AGC CGT CAC GGC AAG CGA CAT GGC AAGAAG TCC AGG 478 Thr Ala Phe Ala Ser Arg His Gly Lys Arg His Gly Lys LysSer Arg 145 150 155 CTG CGC TGC AGC AGA AAG CCT CTG CAC GTG AAT TTT AAGGAG TTA GGC 526 Leu Arg Cys Ser Arg Lys Pro Leu His Val Asn Phe Lys GluLeu Gly 160 165 170 175 TGG GAC GAC TGG ATT ATC GCG CCC CTA GAG TAC GAGGCC TAT CAC TGC 574 Trp Asp Asp Trp Ile Ile Ala Pro Leu Glu Tyr Glu AlaTyr His Cys 180 185 190 GAG GGC GTG TGC GAC TTT CCG CTG CGC TCG CAC CTTGAG CCC ACT AAC 622 Glu Gly Val Cys Asp Phe Pro Leu Arg Ser His Leu GluPro Thr Asn 195 200 205 CAT GCC ATC ATT CAG ACG CTG ATG AAC TCC ATG GACCCG GGC TCC ACC 670 His Ala Ile Ile Gln Thr Leu Met Asn Ser Met Asp ProGly Ser Thr 210 215 220 CCG CCT AGC TGC TGC GTT CCC ACC AAA CTG ACT CCCATT AGC ATC CTG 718 Pro Pro Ser Cys Cys Val Pro Thr Lys Leu Thr Pro IleSer Ile Leu 225 230 235 TAC ATC GAC GCG GGC AAT AAT GTN GTC TAC AAG CAGTAT GAG GAC ATG 766 Tyr Ile Asp Ala Gly Asn Asn Xaa Val Tyr Lys Gln TyrGlu Asp Met 240 245 250 255 GTG GTG GAG TCC TGC GGC TGT AGG TAGCGGTGCTGTCCCGCCAC CTGGGCCAGG 820 Val Val Glu Ser Cys Gly Cys Arg 260 GACCATGGAGGGAGGCCTGA CTGCCGAGAA AGGAGCAGGA GCTGGCCTTG GAAGAGGCCA 880 CAGGTGGGGGACAGCCTGAA AGTAGGAGCA CAGTAAGAAG CAGCCCAGCC TTCCCAGAAC 940 CTTCCAATCCCCCAACCCAG AAGCAGCTAA GGGGTTTCAC AACTTTTGGC CTTGCCAGCC 1000 TGGAAAGACTAGACAAGAGG GATTCTTCTC TTTTTATTAT GGCTTG 1046 263 amino acids amino acidlinear protein 32 Arg Lys Gln Ala Cys Ile Pro Ala Gly Pro Thr Leu ArgGly Ser Ser 1 5 10 15 Gly Thr Gln Pro Arg Pro Ala Gly Lys Ser Phe AspVal Trp Gln Gly 20 25 30 Leu Arg Pro Gln Pro Trp Lys Gln Leu Cys Leu GluLeu Arg Ala Ala 35 40 45 Trp Gly Glu Leu Asp Xaa Gly Asp Thr Gly Ala ArgAla Arg Gly Pro 50 55 60 Gln Gln Pro Pro Pro Leu Asp Leu Arg Ser Leu GlyPhe Gly Arg Arg 65 70 75 80 Val Arg Pro Pro Gln Glu Arg Ala Leu Leu ValVal Phe Thr Arg Ser 85 90 95 Gln Arg Lys Asn Leu Phe Thr Glu Met His GluGln Leu Gly Ser Ala 100 105 110 Glu Ala Ala Gly Ala Glu Gly Ser Cys ProAla Pro Ser Gly Ser Pro 115 120 125 Asp Thr Gly Ser Trp Leu Pro Ser ProGly Arg Arg Arg Arg Arg Thr 130 135 140 Ala Phe Ala Ser Arg His Gly LysArg His Gly Lys Lys Ser Arg Leu 145 150 155 160 Arg Cys Ser Arg Lys ProLeu His Val Asn Phe Lys Glu Leu Gly Trp 165 170 175 Asp Asp Trp Ile IleAla Pro Leu Glu Tyr Glu Ala Tyr His Cys Glu 180 185 190 Gly Val Cys AspPhe Pro Leu Arg Ser His Leu Glu Pro Thr Asn His 195 200 205 Ala Ile IleGln Thr Leu Met Asn Ser Met Asp Pro Gly Ser Thr Pro 210 215 220 Pro SerCys Cys Val Pro Thr Lys Leu Thr Pro Ile Ser Ile Leu Tyr 225 230 235 240Ile Asp Ala Gly Asn Asn Xaa Val Tyr Lys Gln Tyr Glu Asp Met Val 245 250255 Val Glu Ser Cys Gly Cys Arg 260 1345 base pairs nucleic acid singlelinear DNA (genomic) NO NO HUMAN V1-1 CDS 138..1301 mat_peptide990..1301 33 AACTATAGCA CCTGCAGTCC CTGGTCTTGG GTGTAGGGGT GCGCTCCTGGTCCCGCGGCT 60 CAGGGATATG CAGTGACCAA TGGGTTGTTG GCCTGATGGG ACTTTTGGCTTGCTAAACCA 120 AAGCTCGGTT CGGATAG CCC GGG CGA AGA CGT CCG CTG CTC TGGGCC AGG 170 Pro Gly Arg Arg Arg Pro Leu Leu Trp Ala Arg -284 -280 -275CTG GCA GCG TTC AGG CTG GGG CAG AGA CGC GGA GTC GGG CGC TGG CTC 218 LeuAla Ala Phe Arg Leu Gly Gln Arg Arg Gly Val Gly Arg Trp Leu -270 -265-260 CAA CAG GCC TGG CTC CCA CAT CGA AGA CAG CTG GGC CAT TTG CTG TTA 266Gln Gln Ala Trp Leu Pro His Arg Arg Gln Leu Gly His Leu Leu Leu -255-250 -245 GGA GGC CCC GCG CTG ACA GTG TGC AGG ATT TGC TCT TAC ACA GCTCTT 314 Gly Gly Pro Ala Leu Thr Val Cys Arg Ile Cys Ser Tyr Thr Ala Leu-240 -235 -230 TCT CTC TGT CCC TGC CGG TCC CCC GCA GAC GAA TCG GCA GCCGAA ACA 362 Ser Leu Cys Pro Cys Arg Ser Pro Ala Asp Glu Ser Ala Ala GluThr -225 -220 -215 -210 GGC CAG AGC TTC CTG TTC GAC GTG TCC AGC CTT AACGAC GCA GAC GAG 410 Gly Gln Ser Phe Leu Phe Asp Val Ser Ser Leu Asn AspAla Asp Glu -205 -200 -195 GTG GTG GGT GCC GAG CTG CGC GTG CTG CGC CGGGGA TCT CCA GAG TCG 458 Val Val Gly Ala Glu Leu Arg Val Leu Arg Arg GlySer Pro Glu Ser -190 -185 -180 GGC CCA GGC AGC TGG ACT TCT CCG CCG TTGCTG CTG CTG TCC ACG TGC 506 Gly Pro Gly Ser Trp Thr Ser Pro Pro Leu LeuLeu Leu Ser Thr Cys -175 -170 -165 CCG GGC GCC GCC CGA GCG CCA CGC CTGCTG TAC TCG CGG GCA GCT GAG 554 Pro Gly Ala Ala Arg Ala Pro Arg Leu LeuTyr Ser Arg Ala Ala Glu -160 -155 -150 CCC CTA GTC GGT CAG CGC TGG GAGGCG TTC GAC GTG GCG GAC GCC ATG 602 Pro Leu Val Gly Gln Arg Trp Glu AlaPhe Asp Val Ala Asp Ala Met -145 -140 -135 -130 AGG CGC CAC CGT CGT GAACCG CGC CCC CCC CGC GCG TTC TGC CTC TTG 650 Arg Arg His Arg Arg Glu ProArg Pro Pro Arg Ala Phe Cys Leu Leu -125 -120 -115 CTG CGC GCA GTG GCAGGC CCG GTG CCG AGC CCG TTG GCA CTG CGG CGA 698 Leu Arg Ala Val Ala GlyPro Val Pro Ser Pro Leu Ala Leu Arg Arg -110 -105 -100 CTG GGC TTC GGCTGG CCG GGC GGA GGG GGC TCT GCG GCA GAG GAG CGC 746 Leu Gly Phe Gly TrpPro Gly Gly Gly Gly Ser Ala Ala Glu Glu Arg -95 -90 -85 GCG GTG CTA GTCGTC TCC TCC CGC ACG CAG AGG AAA GAG AGC TTA TTC 794 Ala Val Leu Val ValSer Ser Arg Thr Gln Arg Lys Glu Ser Leu Phe -80 -75 -70 CGG GAG ATC CGCGCC CAG GCC CGC GCG CTC GGG GCC GCT CTG GCC TCA 842 Arg Glu Ile Arg AlaGln Ala Arg Ala Leu Gly Ala Ala Leu Ala Ser -65 -60 -55 -50 GAG CCG CTGCCC GAC CCA GGA ACC GGC ACC GCG TCG CCA AGG GCA GTC 890 Glu Pro Leu ProAsp Pro Gly Thr Gly Thr Ala Ser Pro Arg Ala Val -45 -40 -35 ATT GGC GGCCGC AGA CGG AGG AGG ACG GCG TTG GCC GGG ACG CGG ACA 938 Ile Gly Gly ArgArg Arg Arg Arg Thr Ala Leu Ala Gly Thr Arg Thr -30 -25 -20 GCG CAG GGCAGC GGC GGG GGC GCG GGC CGG GGC CAC GGG CGC AGG GGC 986 Ala Gln Gly SerGly Gly Gly Ala Gly Arg Gly His Gly Arg Arg Gly -15 -10 -5 CGG AGC CGCTGC AGC CGC AAG CCG TTG CAC GTG GAC TTC AAG GAG CTC 1034 Arg Ser Arg CysSer Arg Lys Pro Leu His Val Asp Phe Lys Glu Leu 1 5 10 15 GGC TGG GACGAC TGG ATC ATC GCG CCG CTG GAC TAC GAG GCG TAC CAC 1082 Gly Trp Asp AspTrp Ile Ile Ala Pro Leu Asp Tyr Glu Ala Tyr His 20 25 30 TGC GAG GGC CTTTGC GAC TTC CCT TTG CGT TCG CAC CTC GAG CCC ACC 1130 Cys Glu Gly Leu CysAsp Phe Pro Leu Arg Ser His Leu Glu Pro Thr 35 40 45 AAC CAT GCC ATC ATTCAG ACG CTG CTC AAC TCC ATG GCA CCA GAC GCG 1178 Asn His Ala Ile Ile GlnThr Leu Leu Asn Ser Met Ala Pro Asp Ala 50 55 60 GCG CCG GCC TCC TGC TGTGTG CCA GCG CGC CTC AGC CCC ATC AGC ATC 1226 Ala Pro Ala Ser Cys Cys ValPro Ala Arg Leu Ser Pro Ile Ser Ile 65 70 75 CTC TAC ATC GAC GCC GCC AACAAC GTT GTC TAC AAG CAA TAC GAG GAC 1274 Leu Tyr Ile Asp Ala Ala Asn AsnVal Val Tyr Lys Gln Tyr Glu Asp 80 85 90 95 ATG GTG GTG GAG GCC TGC GGCTGC AGG TAGCGCGCGG GCCGGGGAGG 1321 Met Val Val Glu Ala Cys Gly Cys Arg100 GGGCAGCCAC GCGGCCGAGG ATCC 1345 388 amino acids amino acid linearprotein 34 Pro Gly Arg Arg Arg Pro Leu Leu Trp Ala Arg Leu Ala Ala PheArg -284 -280 -275 -270 Leu Gly Gln Arg Arg Gly Val Gly Arg Trp Leu GlnGln Ala Trp Leu -265 -260 -255 Pro His Arg Arg Gln Leu Gly His Leu LeuLeu Gly Gly Pro Ala Leu -250 -245 -240 Thr Val Cys Arg Ile Cys Ser TyrThr Ala Leu Ser Leu Cys Pro Cys -235 -230 -225 Arg Ser Pro Ala Asp GluSer Ala Ala Glu Thr Gly Gln Ser Phe Leu -220 -215 -210 -205 Phe Asp ValSer Ser Leu Asn Asp Ala Asp Glu Val Val Gly Ala Glu -200 -195 -190 LeuArg Val Leu Arg Arg Gly Ser Pro Glu Ser Gly Pro Gly Ser Trp -185 -180-175 Thr Ser Pro Pro Leu Leu Leu Leu Ser Thr Cys Pro Gly Ala Ala Arg-170 -165 -160 Ala Pro Arg Leu Leu Tyr Ser Arg Ala Ala Glu Pro Leu ValGly Gln -155 -150 -145 Arg Trp Glu Ala Phe Asp Val Ala Asp Ala Met ArgArg His Arg Arg -140 -135 -130 -125 Glu Pro Arg Pro Pro Arg Ala Phe CysLeu Leu Leu Arg Ala Val Ala -120 -115 -110 Gly Pro Val Pro Ser Pro LeuAla Leu Arg Arg Leu Gly Phe Gly Trp -105 -100 -95 Pro Gly Gly Gly GlySer Ala Ala Glu Glu Arg Ala Val Leu Val Val -90 -85 -80 Ser Ser Arg ThrGln Arg Lys Glu Ser Leu Phe Arg Glu Ile Arg Ala -75 -70 -65 Gln Ala ArgAla Leu Gly Ala Ala Leu Ala Ser Glu Pro Leu Pro Asp -60 -55 -50 -45 ProGly Thr Gly Thr Ala Ser Pro Arg Ala Val Ile Gly Gly Arg Arg -40 -35 -30Arg Arg Arg Thr Ala Leu Ala Gly Thr Arg Thr Ala Gln Gly Ser Gly -25 -20-15 Gly Gly Ala Gly Arg Gly His Gly Arg Arg Gly Arg Ser Arg Cys Ser -10-5 1 Arg Lys Pro Leu His Val Asp Phe Lys Glu Leu Gly Trp Asp Asp Trp 510 15 20 Ile Ile Ala Pro Leu Asp Tyr Glu Ala Tyr His Cys Glu Gly Leu Cys25 30 35 Asp Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn His Ala Ile Ile40 45 50 Gln Thr Leu Leu Asn Ser Met Ala Pro Asp Ala Ala Pro Ala Ser Cys55 60 65 Cys Val Pro Ala Arg Leu Ser Pro Ile Ser Ile Leu Tyr Ile Asp Ala70 75 80 Ala Asn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met Val Val Glu Ala85 90 95 100 Cys Gly Cys Arg 17 base pairs nucleic acid single linearDNA (genomic) NO NO primer number 8 35 TGTATGCGAC TTCCCGC 17

What is claimed is:
 1. A DNA molecule comprising an isolated DNAsequence encoding a BMP-12 related protein.
 2. A DNA molecule accordingto claim 1, wherein said DNA sequence is selected from the groupconsisting of: (a) nucleotides #496, #571 or #577 to #882 of SEQ IDNO:1; (b) nucleotides #605 or #659 to #964 of SEQ ID NO:25; and (c)sequences which hybridize to (a) or (b) under stringent hybridizationconditions and encode a BMP-12 related protein which exhibits theability to form tendon/ligament-like tissue.
 3. A DNA moleculecomprising the DNA sequence of claim 1 wherein said DNA sequence isselected from the group consisting of: (a) nucleotides encoding foramino acids #-25, #1 or #3 to #104 of SEQ ID NO:2; (b) in a 5′ to 3′direction, nucleotides encoding a propeptide selected from the groupconsisting of native BMP-12 propeptide and a BMP protein propeptide; andnucleotides encoding for amino acids #-25, #1 or #3 to #104 of SEQ IDNO:2; and (c) nucleotides encoding for amino acids #1 or #19 to #120 ofSEQ ID NO:26; (d) in a 5′ to 3′ direction, nucleotides encoding apropeptide selected from the group consisting of native BMP-12propeptide and a BMP protein propeptide; and nucleotides encoding foramino acids #1 or #19 to #120 of SEQ ID NO:26; (e) sequences whichhybridize to any of (a) through (d) under stringent hybridizationconditions and encode a BMP-12 related protein which exhibits theability to form cartilage and/or bone.
 4. A host cell transformed with aDNA molecule according to claim
 1. 5. A host cell transformed with theDNA molecule of claim
 2. 6. A host cell transformed with the DNAmolecule of claim
 3. 7. An isolated DNA molecule having a sequenceencoding a BMP-12 protein which is characterized by the ability toinduce the formation of tendon/ligament-like tissue, said DNA moleculecomprising a DNA sequence selected from the group consisting of: (a)nucleotide #496, #571 or #577 to #882 of SEQ ID NO:1; (b) nucleotide#605 or #659 to #964 of SEQ ID NO:25; and (c) naturally occurringallelic sequences and equivalent degenerative codon sequences of (a) or(b).
 8. A host cell transformed with the DNA molecule of claim
 7. 9. Avector comprising a DNA molecule of claim 7 in operative associationwith an expression control sequence therefor.
 10. A host celltransformed with the vector of claim
 9. 11. A method for producing apurified BMP-12 protein, said method comprising the steps of: (a)culturing a host cell transformed with a DNA molecule according to claim2, comprising a nucleotide sequence encoding a BMP-12 related protein;and (b) recovering and purifying said BMP-12 related protein from theculture medium.
 12. A method for producing a purified BMP-12 relatedprotein said method comprising the steps of: (a) culturing a host celltransformed with a DNA molecule according to claim 3, comprising anucleotide sequence encoding a BMP-12 related protein; and (b)recovering and purifying said BMP-12 related protein from the culturemedium.
 13. A method for producing a purified BMP-12 related proteinsaid method comprising the steps of: (a) culturing a host celltransformed with a DNA molecule according to claim 7, comprising anucleotide sequence encoding a BMP-12 related protein: and (b)recovering and purifying said BMP-12 related protein from the culturemedium.
 14. A purified polypeptide comprising an amino acid sequenceselected from the following group: (a) from amino acid #-25 to aminoacid #104 as set forth in SEQ ID NO:2; (b) from amino acid #1 to aminoacid #104 as set forth in SEQ ID NO:2. (c) from amino acid #3 to aminoacid #104 as set forth in SEQ ID NO:2. (d) from amino acid #1 to aminoacid #120 as set forth in SEQ ID NO:26; and (d) from amino acid #19 toamino acid #120 as set forth in SEQ ID NO:26.
 15. A purified polypeptidewherein said polypeptide is in the form of a dimer comprised of twosubunits, each with the amino acid sequence of claim
 14. 16. A purifiedprotein produced by the steps of (a) culturing a cell transformed with aDNA molecule comprising the nucleotide sequence from nucleotide #496,#571 or #577 to #882 as shown in SEQ ID NO:1; and (b) recovering andpurifying from said culture medium a protein comprising the amino acidsequence from amino acid #-25, amino acid #1 or amino acid #3 to aminoacid #104 as shown in SEQ ID NO:2.
 17. A purified BMP-12 related proteincharacterized by the ability to induce the formation oftendon/ligament-like tissue.
 18. A pharmaceutical composition comprisingan effective amount of the BMP-12 related protein of claim 17 inadmixture with a pharmaceutically acceptable vehicle.
 19. A method forinducing tendon/ligament-like tissue formation in a patient in need ofsame comprising administering to said patient an effective amount of thecomposition of claim
 18. 20. A pharmaceutical composition fortendon/ligament-like tissue healing and tissue repair said compositioncomprising an effective amount of the protein of a BMP-12 relatedprotein in a pharmaceutically acceptable vehicle.
 21. A method fortreating tendinitis, or other tendon or ligament defect in a patient inneed of same, said method comprising administering to said patient aneffective amount of the composition of claim
 20. 22. A chimeric DNAmolecule comprising a DNA sequence encoding a propeptide from a memberof the TGF-β superfamily of proteins linked in correct reading frame toa DNA sequence encoding a BMP-12 related polypeptide.
 23. A chimeric DNAmolecule according to claim 22, wherein the propeptide is the propeptidefrom BMP-2.
 24. A heterodimeric protein molecule comprising one monomerhaving the amino acid sequence of the polypeptide of claim 14, and onemonomer having the amino acid sequence of a protein of the TGF-βsuperfamily.
 25. A method for inducing tendon/ligament-like tissueformation in a patient in need of same comprising administering to saidpatient an effective amount of a composition comprising a proteinencoded by a DNA sequence selected from the group consisting of: (a)nucleotides #496, #571 or #577 to #882 of SEQ ID NO:1; (b) nucleotides#845 or #899 to #1204 of SEQ ID NO:3; (c) nucleotides #605 or #659 to#964 of SEQ ID NO:25; and (d) sequences which hybridize to (a), (b) or(c) under stringent hybridization conditions and encode a protein whichexhibits the ability to form tendon/ligament-like tissue.
 26. A methodfor inducing tendon/ligament-like tissue formation in a patient in needof same comprising administering to said patient an effective amount ofthe composition comprising a tendon/ligament-like tissue inducingprotein having an amino acid sequence selected from the group consistingof: (a) amino acids #-25, #1 or #3 to #104 of SEQ ID NO:2; (b) aminoacids #1 or #19 to #120 of SEQ ID NO:4; (c) amino acids #1 or #19 to#120 of SEQ ID NO:26; and (d) mutants and/or variants of (a), (b) or (c)which exhibit the ability to form tendon and/or ligament.
 27. Apharmaceutical composition for tendon/ligament-like tissue repair, saidcomposition comprising an effective amount of a BMP-12 related proteinin a pharmaceutically acceptable vehicle.
 28. A method for treatingtendinitis, or other tendon or ligament defect in a patient in need ofsame, said method comprising administering to said patient an effectiveamount of the composition of claim 27.